A. MAYER, G. B. ZONDAG and L.A.M. HENSGENS
MOLBAS Research Group, Department of Plant Molecular Biology, Leiden University, Wassenaarseweg 64, 2333AL Leiden, The Netherlands
Transformation of rice, like other cereals, is a difficult task (see for a review: Potrykus 1990). Most methods, such as protoplast electroporation (Shimamoto et al. 1989), polyethylene glycol (PEG) treatment (Zhang and Wu 1988; Hayashimoto et al. 1990) and particle bombardment involve much tissue culture work.
Methods for detecting transformed tissue employ phenotypic expression of resistance or reporter genes or are based on Southern analysis of purified rice DNA. The expression of the gene and tight correlation with physical data is considered to be proof of the transformed state (e.g. Potrykus) of the tissue or whole plant. Since expression of many genes (including chimeric) is under developmental control, actually transformed tissue may be considered as nontransgenic and disposed of (Benfey and Chua 1989; Meijer et al. 1991).
Most vectors used for transformation of rice harbor the hygromycin phosphotransferase (HPT) gene for selection and the β-D-glucuronidase reporter gene. We have optimized the Polymerase Chain Reaction (PCR) method using three primer pairs in one reaction. By this multiplex PCR analysis we were able to detect an HPT-gene, a GUS-gene fragment and a fragment derived from the single copy gene Gos5 (de Pater et al. 1990). The latter was chosen as an internal control for the quality of the PCR analysis.
To minimize labour, an easy and siniple method for DNA isolation was developed.
With help of the OLIGOTM program (copyright 1988-1990 Wojciech Rychlik) primer-pairs with high melting temperatures (Tm), minimal self-complementarity and high specificity to the target DNA were selected. Tm and lenghts of the three primer pairs were chosen so that multiplex PCR was possible (see Table 1). The lengths of the expected GUS-, HPT- and Gos5-fragments were 683, 419 and 231 bp, respectively.
Using CsCl purified plasmid (construct HH271, containing the GUS-and HPT-gene) and genomic DNA isolated from rice tissue transgenic with HH271 (Meijer et al. 1991) and a genomic lambda clone carrying the Gos5-gene (de Pater et al. 1990), PCR amplification was optimized for the three primer sets seperately. When three individual primer pairs were annealed at 72°C, the background was minimal (data not shown).
Different dilutions of plasmid and phage DNA were used to optimize the concentrations of MgCl2, Tween 20, BSA, dNTP's, primers and the amount of AmpliTaq. With the optimized protocol, one femtogram of plasmid or phage DNA was sufficient to obtain visible bands (data not shown). Hence a mixture of all 3 primer pairs was used in a multiplex PCR to study whether amplification of the three fragments could be performed in a single tube. Different quantities (10, 1 and 0.1 ng) of genomic DNA from HH271 transformed rice (cv. Taipei 309) cell suspension were tested (see Fig. 1).
Table 1 - Primer sequences for detection of the GUS-, HPT- and Gos 5-gene. The position within the gene after the AUG start codon is given in nucleotides. Melting temperatures Tm(°C) were calculated using a primer concentration of 250 pM and a salt concentration of 50 mM.
primer primer sequence position Tm within gene (°C) after AUG GUS-upper 5'CAGCGAAGAGGCAGTCAACGGGGAA 3' 1124 67.7 GUS-lower 5'CATTGTTTGCCTCCCTGCTGCGGTT 3' 1807 67.4 HPT-upper 5'CGCACAATCCCACTATCCTTCGCAA 3' -94 64.6 HPT-lower 5'GGCAGTTCGGTTTCAGGCAGGTCTT 3' 325 64.3 Gos5-upper 5'CGACCTCGAGGACATCGGCAACACC 3' 292 68.0 Ges5-lower 5'GCCGAGCAGCAGGAACTTGAGCAGG 3' 523 67.7
Optimized PCR-protocol: 100 microM ddATP, ddCTP, ddGTP, ddTTP 2 mM MgCl2 25 mM KCl 20 mM TRIS-HCl pH 8.3 0.5% Tween 20 (Sigma) deionized, filter sterilized 0.05 mg acetylated BSA, Promega, USA 10 microM primers GUS-upper, GUS-lower 10 microM primers HPT-upper, HPT-lower 2.5 microM primers Gos5-upper, Gos5-lower 2 microl DNA x microl H2O up to a volume of 90 microl at 85°C: 1 unit AmpliTaqTM (Perkin Elmer Cetus)in buffer (20 mM TRIS-HCl pH 8.3, 25 mM KCl, 0.05 mg acetylated BSA, 0.5% Tween 20, H2O up to a volume of 10 microl), reaction volume 100microl overlaid with 70microl mineral oil PCR-program: (run in PREMTM III, LEM Scientific) 20 min. 98°C cooling to 85°C addition of 1 u AmpliTaq per reaction 45 cycles: 1 min. 93°C 2 min. 72°C 5 min. 72°C
Fig. 1. Influence of addition of 1 u AmpliTaq enzyme before (lanes B) and after (lanes A) the first denaturation step. The numbers above the slots refer to the amount of genomic rice (cv. T309) DNA, which was isolated from 3-5 g cell suspension transgenic for plasmid HH271 according to Hensgens and van OsRuygrok (1989). 45 PCR cycles were performed as following: One min. 93°C and 2 min. 72°C. Concentration of all 6 primers was 10μM. Ten μl of the PCR reaction (100μl) were loaded on a 2% agarose gel containing 0.5μg/ml ethidium bromide, and the DNA fragments fractionated by electrophoresis.
Fig. 2. Determination of the optimal volume of DNA extract for amplification. Various volumes of extraxt (5, 2, 0.5 μl) were added as indicated to the PCR reaction mixture and amplified with 1U AmpliTaq added in the hot-start procedure run for 45 cycles: One min. at 93°C and 2 min. at 72°C. Primer concentrations were 10μM for the primer pairs of the GUS and HPT genes and 2.5μM for Gos5. 10μl of the 100μl reaction volume was loaded on a 2% agarose gel containing 0.5μg/ml ethidium bromide and electrophorized.
One unit of AmpliTaq was added either at room temperature before the first denaturation step of 93°C or at 85°C after an initial denaturation step of 1 minute at 93°C (hot-start). In both cases no previous heat-treatment at 98°C was made.
For the amplification of all 3 fragments using the hotstart procedure 0.1 ng of genomic DNA was sufficient (Figure 1). The size of the rice genome is estimated to be approximately 9 X 108 bp (Bennett and Smith 1976). Therefore, 0.1 ng rice DNA is equivalent to 100 genome copies, thus proving the sensitivity of the reaction.
Using the sensitive PCR protocol several DNA isolation methods were tested. It appeared that low amounts of EDTA, SDS, cysteine and β-mercaptoethanol did not interfere with the PCR amplification. A reliable, efficient and fast method for preparing samples was found to be:
50 mg of fresh plant tissue (root, callus or green tissue), in Potter tube (Kontes Scientific Glassware/Instruments, Vineland New Jersey) 200 μl of isolation buffer: 200 mM TRIS-HCl pH 8.3 20 mM Na-EDTA 500 mM NaCl 0.1% cysteine 20 mM β-mercaptoethanol 0.2% sodium-dodecyl sulfate (SDS) 100 μg/ml proteinase K homogenization for 1 minute with a pestle (Kontes Scientific Glassware/Instruments) in an electric driven Potters homogenizer (B. Braun, Melsungen, FRG) incubation for 6 hours at 65°C 1 hour at 105°C spinning down in Eppendorf centrifuge for 1 min. supernatant was used for PCR analysis.
None of the compounds in the extraction buffer inhibited the activity of AmpliTaq in the used concentrations. In this way a sample was prepared from 50 mg of transgenic callus transformed with HH271 (Meijer et al. 1991). Different amounts of extract were tested (Fig. 2).
It can be seen from Figure 2 that 2 μl of extract was sufficient to yield clear bands of the 3 fragments. When it is estimated that 1% of the 5 million rice callus cells (50 mg) released their genomic DNA, this quantity should be equivalent to 500 genome copies. This number is in the same order of magnitude as the numbers found employing plasmid or purified genomic DNA.
Both the optimized PCR and the DNA extraction method therefore fulfill the requirement of being simple and quick and allow the screening of large numbers of samples like calli or parts of rice plants. Nevertheless the PCR analysis is no proof for stable integration of a foreign gene into the genome.
As we found optimum temperatures for annealing and denaturation to be critical, optimization of temperatures for each type of thermocycler should be carried out. Using a thermosensor we found rather large (2-4°C) differences in different PCR apparatus at the same temperature settings. Secondly, as optimal amplification of fragments in the PCR reaction depends on the concentration of template DNA, the absence of amplified fragments does not exclude its presence. It can be present in too high or too low a concentration. Therefore, false negatives can not always easily be discriminated from truely nontransformed tissue. As a routine we further incorporate negative controls to check contamination reagents for the PCR reaction, DNA isolation and rice tissue culture medium since the PCR is known to be very sensitive to contaminating DNA. It is known that using 45 cycles increases the risk of amplifying contaminating DNA molecules. We have found that using 35 cycles minimizes this risk and hardly any false positives have been found.
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