Rice is the most important cereal of the developing world and a model
crop plant. A relatively efficient method to transform rice using Agrobacterium
has already been widely used to generate transgenic plants (Hiei et
al., 1994; Datta et al., 1996; Roy and Wu, 2001). This method
has also been used in functional genomics studies. Recently, draft sequences
of the entire rice genome have been published. To assign functions to
the assumed 50,000 rice genes, a rice gene-disruption library needs to
be constructed. To achieve a 99% probability of tagging all the genes
in rice genome, with an average gene size of 3 kb, 660,000 randomly-produced
gene-disruption plant lines are required (Jeon et al., 2000).
We therefore tested several modifications of the standard protocol in
an effort to improve the efficiency of the Agrobacterium-mediated
transformation procedure for rice, in terms of reducing the effort and
the time required to produce a large number of transgenic plants. Enriquez-Obregon
et al. (1999) used three antinecrotic compounds to treat rice explants
and successfully produced transgenic plants. Their system uses a non-supervirulent
Agrobacterium strain and can also omit the use of acetosyringone,
which induces vir genes. Their aim was to show that the antinecrotic compounds
could reduce the difficulty of Agrobacterium infection in monocots
by preventing necrosis of target cells. In our case, however, we used
these compounds (40 mg/L L-cysteine, 5 mg/L silver nitrate and 15 mg/L
ascorbic acid) in addition to a supervirulent Agrobacterium strain
carrying a binary vector and acetosyringone to enhance the frequency of
transgenic plant recovery using calli. We made use of these three antinecrotic
compounds at a minimal level as optimized by Enriquez-Obregon et al.
(1999) during co-cultivation of the embryogenic calli with Agrobacterium
cells. We also altered the duration for selection and regeneration.
Four-week-old actively-growing mature-seed-derived calli of the japonica
variety Nipponbare were infiltrated under vacuum for 15-20 minutes with
Agrobacterium cells resuspended in liquid MS medium supplemented
with 500 mg/L casein hydrolysate, 68.5 g/L sucrose, 36 g/L glucose and
200 microM acetosyringone. This was followed by a 3-day co-cultivation
in the dark of the infiltrated calli on solid MS medium (pH 5.2) supplemented
with 2 mg/L 2,4-D, 10 g/L glucose, 30 g/L sucrose, 100 microM acetosyringone,
40 mg/L L-cysteine, 15 mg/L ascorbic acid, 5 mg/L silver nitrate. The
treated calli were then rinsed with sterile distilled water containing
250 mg/L cefotaxime, and the excess water was soaked onto sterile filter
papers before plating the calli for the first selection cycle in the dark
using the same medium used for callus induction but supplemented with
50 mg/L hygromycin and 250 mg/L cefotaxime. After 15-18 days, calli were
transferred to regeneration selection medium, which is MS medium supplemented
with 2 mg/L kinetin, 0.5 mg/L alpha-naphthalene acetic acid (NAA) and
same concentrations of the antibiotics. A total of three 3-week regeneration
selection cycles were carried out under 14/10 hours light/dark photoperiods.
Cefotaxime was omitted in the second cycle of regeneration and onwards.
Thus the total time to regenerate transgenic plants was 11 weeks after
co-cultivation with Agrobacterium cells. Next, the plantlets were
grown in tap water for 7 days for acclimatization and for fast rooting,
before transferring them to soil under greenhouse conditions. In our previous
protocol, we used 3 selection cycles in the dark (2-3 weeks each) and
2 regeneration cycles (3-4 weeks each) with the total time of approximately
15 weeks.
For transformation, we used a plasmid carrying the GUS reporter gene and
the hygromycin phosphotransferase gene as the selectable marker, both
driven by the constitutive CaMV 35S promoter. The plasmid was mobilized
into Agrobacterium strain LBA4404. We repeated each experiment
3 times, each time regenerating approximately 100-150 plants. In the different
experiments, 90-95% of the regenerated plants tested positive for GUS
in a histochemical assay. The results on the presence of the transgene
were confirmed by Southern blot hybridization.
In the preliminary experiments, we tried three different schedules for
treating the calli with the antinecrotic compounds. We used a 30-minute
pre-treatment with the antinecrotic compounds for one batch of calli,
treatment only during co-cultivation with Agrobacterium cells for
the second batch of calli and a combination of the two for a third batch
of calli. A fourth control batch of calli was transformed at the same
time without any treatment by antinecrotic compounds. The preliminary
results showed that using the antinecrotic compounds only during co-cultivation
gave the best results and it did not require an additional step of pretreatment.
Thus, we used this schedule for the next two experiments.
Overall, we found that the number of putative independent transgenic regenerants
was increased by 32-42% when calli were treated with antinecrotic compounds
as compared to the corresponding control set (Table 1). It is likely that
these compounds, with known anti-oxi-
dant activity, helped to minimize the damage due to oxidative bursts
during plant-microorganism interaction and to prevent necrosis of rice
cells. These treatments resulted in a better survival rate of the calli,
thereby increasing the overall frequency of transgenic plant regeneration.
For our experiments, approximately 90 GUS-positive plants were randomly
chosen to perform Southern blot analyses, 40% of which were found to carry
a single copy of the transgene. All the plants regenerated by this method
had normal seed setting and grain filling as compared to the control set
of transgenic plants. As shown in Table 1, the transformation frequencies
were 54 and 70% in two independent experiments. The regeneration frequency
is much higher even in the control plants (53 and 38%, Table 1) that were
not treated with antinecrotic compounds as compared to the previous reports
of 3-8% in a japonica variety TNG67 (Roy and Wu, 2001). It appears
that the difference in regeneration frequency is also attributed to the
alterations in the conditions of the selection cycles. In addition, variations
in the rice cultivar used and minor differences in cell culture conditions
can affect the overall regeneration efficiency between independent experiments.
In conclusion, in the present experiment, we have achieved a large increase
in the regeneration frequency of transgenic rice in a shorter amount of
time. Thus, the improved method would greatly favor any experiment that
requires large-scale transgenic rice production such as those carried
out in functional genomics studies.
References
Datta, K., N. Oliva, L. Torrizo, E. Abrigo, G. S. Khush and S. K. Datta,
1996. Genetic transformation of Indica and Japonica rice by Agrobacterium.
Rice Genetics Newsletter 13: 136-139.
Enriquez-Obregon, G. A., D. L. Prieto-Samsonov, G. A. de la Riva, M. Perez,
G. Selman-Housein and R. I. Vazquez-Padron, 1999. Agrobacterium-mediated
japonica rice transformation: a procedure assisted by antinecrotic treatment.
Plant Cell, Tissue and Organ Culture 59: 159-168.
Hiei, Y., S. Ohta, T. Komari and T. Kumashiro, 1994. Efficient transformation
of rice (Oryza sativa L.) mediated by Agrobacterium and
sequence analysis of boundaries of T-DNA. Plant J. 6: 271-282.
Jeon, J. S. et al., 2000. T-DNA insertional mutagenesis for functional
genomics in rice. Plant J. 22: 561-570.
Roy, M. and R. Wu, 2001. Arginine decarboxylase transgene expression and
analysis of environmental stress tolerance in transgenic rice. Plant Science
160: 869-875.
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