- Email: [email protected]
[37] Selectable markers for rice transformation
426
REPORTER GENES
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9. Transblot for 3-4 hr at 40 V; make sure that the nitrocellulose is toward the anode. 10. Incubate the filter for 2-4hr at room temperature or overnight at 4° in PBS-ovalbumin buffer [150 mM NaCI, 12.6 mM NaHzPO 4 • 2H20, 14 mM K H z P O 4, pH 7.4, 0.5% (w/v) ovalbumin] with rocking. 11. Soak the filter at room temperature for 2 hr with gentle shaking in anti-PAT antibody solution. 12. Wash three times in PBS containing 0.1% (v/v) Triton X-100 for 10 min. 13. Incubate for 2 hr at room temperature in a second antibody solution (alkaline phosphatase-conjugated anti-rabbit IgG; Sigma). 14. Wash once with PBS [with 0.1% (v/v) Triton X-100] for 30 min and three times with PBS for 30 min. 15. Add freshly made staining solution [30 mg p-nitro blue tetrazolium chloride (NBT; Bio-Rad) in 1 ml 70% (v/v) N,N-dimethylformamide, 15 mg 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP; Sigma) in 1 ml 100% N,N-dimethylformamide, 98 ml carbonate buffer (0.1 M NaHCO 3 + 1.0 mM MgCI2) (pH 9.8)] and shake until the reaction product is visible. 16. Rinse the filter in distilled water and dry between two sheets of Whatman 3MM paper in the dark.
[37] Selectable Markers for R i c e T r a n s f o r m a t i o n By
A L L A N C A P L A N , R U D Y DEKEYSER,
and MARC V A N MONTAGU
Introduction
Choosing Convenient Method to Introduce D N A A number of techniques have been developed to transfer DNA into rice. Some techniques, such as polyethylene glycol (PEG)-mediated or electroporation-mediated gene transfer, have been used repeatedly by several groups, 1-9 and are becoming well-established routines for genetic I K. Toriyama, Y. Arimoto, H. Uchimiya, and K. Hinata, Bio/Technology 6, 1072 (1988). 2 H. M. Zhang, H. Yang, E. L. Rech, T. J. Golds, A. S. Davis, B. J. Mulligan, E. C. Cocking, and M. R. Davey, Plant Cell Rep. 7, 379 (1988). 3 K. Shimamoto, R. Terada, T. Izawa, and H. Fujimoto, Nature (London) 338, 274 (1989). 4 R. Matsuki, H. Onodera, T. Yamauchi, and H. Uchimiya, Mol. Gen. Genet. 220, 12 (1989). 5 S. K. Datta, A. Peterhans, K. Datta, and I. Potrykus, Bio/Technology 8, 736 (1990).
METHODS IN ENZYMOLOGY,VOL. 216
Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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SELECTABLE MARKERS FOR RICE TRANSFORMATION
TABLE I REPRESENTATIVE TRANSFORMATION EFF1CIENCIES OF RICE PROTOPLASTS DNA Selectable marker~
concentration (/xg/ml)
P35S:nptlI P35S:nptlI P35S:hpt P35S:hpt P35S:hpt Pnos:hpt P35S:hpt P35S:nptlI P35S:nptlI P35S:hpt
16 20 10 1 1.3 40 40 50 75 20
Transformation frequency (10 -6)
Estimated
introduction b
Selective agent (/zg/ml) c
copy number
Ref.
E E E E P P P P P E
G418 (20) Kan (100) Hyg (20) Hyg (50) Hyg (25) Hyg (50, 100) Hyg (50, 100) Kan (100) Kan ( 2 0 0 ) Hyg (50)
4 280 Up to 6500 120-200 5-44 d 76; 59 197; 176 20 3250-6500 d 134-137
1-2 1-2 2-10 ->3 50-100 -1-10 2-10 20-30 1-10
1 2 3 4 5 6 6 7 8 9
Method of
'~ P35S, cauliflower mosaic virus (CaMV) promoter; Pnos, nopaline synthase promoter; nptlI, neomycin phosphotransferase II gene; hpt, hygromycin phosphotransferase gene. b E, Electroporation; P, PEG. " Kan, Kanamycin; Hyg, hygromycin. d Indica variety of rice; all other experiments use japonica varieties.
engineering projects. Others including pollen tube transformation, ~0particle guns, 1~and Agrobacterium tumefaciens, 12which have been introduced more recently, involve more specialized skills, and hence seem more difficult to apply repeatedly. Genes transferred by any of the first four methods mentioned rely on host D N A repair systems to link the foreign genes to the chromosomes. The integration sites may be distributed at random along the sequences of the introduced DNA, and thus may disrupt the gene being transferred. However, Agrobacterium and its tumor-inducing (Ti) plasmid-mediated system of cell transformation should be able to transfer D N A in a defined manner producing predictable end points for integration. Electroporation and PEG-mediated gene transfer do not differ greatly either in terms of efficiency or ease of application (Table I). Each functions 6 A. Hayashimoto, Z. Li, and N. Murai, Plant Physiol. 93, 857 (1990). 7 A. Peterhans, S. K. Datta, K. Datta, G. J. Goodall, I. Potrykus, and J. Paszkowski, Mol. Gen. Genet. 222, 361 (1990). 8 j. Peng, L. J. Lyznik, L. Lee, and T. K. Hodges, Plant Cell Rep. 9, 168 (1990). 9 y . Tada, M. Sakamoto, and T. Fujimura, Theor. Appl. Genet, 80, 475 (1990). z0 Z.-X. Luo and R. Wu, Plant Mol. Biol. Rep. 6, 165 (1988). tl M. E. Fromm, F. Morrish, C. Armstrong, R. Williams, J. Thomas, and T. M. Klein, Bio/ Technology 8, 833 (1990). 12 D. M. Raineri, P. Bottino, M. P. Gordon, and E. W. Nester, Bio/Technology 8, 33 (1990).
428
REPORTER GENES
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by opening pores through which DNA can diffuse into the cell, and eventually into the nucleus, where transcription is possible. Both of these methods require similar amounts of purified DNA (generally ranging from 10 to 40 ~g/sample). For the sake of convenience, it is advisable to maintain the genes that are to be transferred on high-copy number, stably segregating Escherichia coli vectors such as those derived from the modified ColE1 origin present in plasmids such as pUC18. ~3 Whereas it has been demonstrated that plasmids as large as 200 kilobases (kb) can be electroporated into protoplasts, 14it is prudent to keep in mind that larger plasmids often yield less DNA during isolation from bacteria. One way of circumventing size constraints is to separate the sequences to be transferred into two sets. For example, the selected marker and the gene under investigation may be cloned on individual vectors and mixed prior to DNA transfer. Molar ratios of 1 : 2, 1 : 1, or 2 : 1 (selected-unselected) yield similar frequencies of cotransformation that differ as much between replicates as between planned variations ( 1 4 - 8 5 % ) . 4,7,9 For some experiments such two-component systems may offer several advantages. First, the cotransformed vector may contain the same restriction sites or expression sequences present in the selected marker without complicating the cloning strategy or increasing the risk of homology-dependent deletion formation that can occur between repeated sequences in the same bacteria. Second, the selected marker and the nonselected gene can integrate at independent loci and thus segregate during meiosis. In this way, primary transformants can produce descendants that are no longer drug resistant, yet retain the unselected gene of interest. Such plants thus have very little extraneous DNA, and can be retransformed with the same selectable marker, should later experiments require the introduction of other genes.
Materials
Protoplast and Plant Culture Media and Solutions Linsmaier and Skoog (LS) medium15:1 liter of deionized water, 4.4 g of Murashige and Skoog salts (Sigma Chemical Co., St. Louis, MO), 1 mg of thiamin hydrochloride, 100 mg of myo-inositol, 30 g of sucrose, 4 g of agarose, pH 5.7. Autoclave, cool to 55°, and add 2 mg of 2,4dichlorophenoxyacetic acid (2,4-D) dissolved in dimethyl sulfoxide (DMSO; from a stock of 10 mg/ml) 13 C. Yanisch-Perron, J. Vieira, and J. Messing, Gene 33, 103 (1985). 14 W° H. R. Langridge, B. J. Li, and A. A. Szalay, Plant Cell Rep. 4, 355 (1985). 15 E. M. Linsrnaier and F. Skoog, Physiol. Plant. 18, 100 (1965).
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429
CPW13M16:1 liter of deionized water, 101 mg of KNO3, 27.2 mg of KHzPO 4, 1480 mg of CaC12- 2H20, 0.025 mg of MgSO4" 7H20, 0.16 mg of KI, 0.025 mg of CuSO4.5H20, 975 mg of 2-(N-morpholino) ethanesulfonic acid, 130 g of mannitol, pH 5.8. Autoclave KpR (per liter of deionized H20)17: As this medium is quite complex, it is advisable to make several stock solutions. Macronutrients can be added as dry powder: 1900 mg of KNO 3, 600 mg of NH4NO 3, 600 mg of CaCI z • 2 H 2 0 , 300 mg of M g S O 4 • 7 H 2 0 , 300 mg of KCI, 170 mg of KHzPO 4. Micronutrients are added as 10 ml of a I00 x stock solution containing (per liter) 1000 mg of MnSO 4 • HzO, 300 mg of H3BO3,200 mg of Z n S O 4 • 7 H 2 0 , 75 mg of KI, 25 mg of N a 2 M o O 4 • 2H20, 2.5 mg o f C u S O 4 • 5 H 2 0 , and 2.5 mg of CoC1z • 6H20. Organic acids are added as 10 ml of a I00 x stock solution containing (per liter) 1 g of citric acid, 1 g of malic acid, 1 g of fumaric acid, and 0.5 g of sodium pyruvate. Adjust pH of stock solution to 5.5 with NH4OH. Several vitamins are added as 10 ml of a 100 x stock containing (per liter) 100 mg of nicotinamide, 100 mg of ascorbic acid, 100 mg of pyridoxinhydrochloride, 100 mg of thiamin hydrochloride, 50 mg of calcium pantothenate, 50 mg of choline chloride, 20 mg of folic acid, and 10 mg of riboflavin. KpR medium is completed by addition of 100 mg of m y o inositol (as dry powder), 10/~1 ofp-aminobenzoic acid (1 mg/ml) stock in H 2 0 ) , 10 /zl of vitamin A (0.5 mg/ml stock in ethanol), 10/zl of vitamin D3 (0.5 mg/ml stock in ethanol), 10/zl of vitamin B12 (1 mg/ ml stock in ethanol), and 1.0 ml of biotin (0.5 mg/ml stock in water). Add 1.25 g of from a dry mix consisting of D-sucrose (10 g), D-fructose (5 g), D-ribose (5 g), D-xylose (5 g), D-mannose (5 g), D-cellobiose (5 g), L-rhamnose (5 g), D-mannitol (5 g), D-sorbitol (5 g). Iron is supplied to the medium as FeSO4" 7H20 (28.75 mg) and Na 2 EDTA (37.25 mg). In our experiments we use 10% (w/v) glucose as osmoticum, omit the casamino acids and the coconut water, and use 0.5 mg of 2,4-D (10 mg/ml stock in DMSO), 1 mg of naphthaleneacetic acid (10 mg/ml stock in DMSO), and 0.5 mg of zeatin (5 mg/ml stock in DMSO) as hormones. The final pH of the solution should be 5.7. In general, the medium is filter sterilized using a 0.2-~m filter unit (Nalgene, Nalge, Rochester, NY), and stored at 4 °. To make agarose for embedding protoplasts, use deionized water to prepare a 2% Seaplaque agarose (FMC, Rockland, ME) solution, autoclave, cool to 45 °, and mix with an equal volume of 2 × KpR solution prior to use 16 E. M. Frearson, J. B. Power, and E. C. Cocking, Dev. Biol. 33, 130 (1973). 17 K. N. Kao, Mol. Gen. Genet. 150, 225 (1977).
430
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Electroporation buffer (EPR): 10 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), pH 7.2, 4 mM CaCI2, 10% (w/v) glucose
Solutions for NPTII Assay Use distilled water for all solutions and buffers. Extraction buffer (4 × ): 100 mM Tris-HC1 (pH 6.8), 0.3 mg/ml leupeptin, 2% (v/v) 2-mercaptoethanol Loading buffer (10 × ): 50% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol, 0.5% (v/v) sodium dodecyl sulfate (SDS), 0.005% (v/v) bromphenol blue Separation gel: For 30 ml, 10 ml of 30% (w/v) acrylamide, 3.9 ml of 2% (w/v) bisacrylamide, 11.2 ml of 1 M Tris-HCl (pH 8.7), 4.5 ml of water. When ready to pour gel, add 0.2 ml of 10% (w/v) ammonium persulfate (APS) and 0.04 ml of N,N,N',N'-tetramethylethylenediamine) (TEMED) Stacking gel: For 10 ml, 1.67 ml of 30% (w/v) acrylamide, 1.3 ml of 2% (w/v) bisacrylamide, 1.25 ml of 100 M Tris-HC1 (pH 6.8), 5.6 ml of water. When ready to pour, add 0.1 ml of 10% (w/v) APS and 0.01 ml of TEMED Running buffer: 6 g/liter Tris, 14.9 g/liter glycine (do not adjust pH) Reaction buffer: 50 mM Tris-HC1 (pH 7.5), 200 mM NH4C1, 25 mM MgC12, 0.5 mM dithiothreitol (DTT) Washing buffer: 100 mM Na2HPO 4 (pH 7.0; adjusted with HC1) Methods and Discussion
Possible Selective Agents The efficiency of any selection technique is determined both by the ability of the selective agent to inhibit untransformed cells, and by the ability of the detoxifying gene to protect transformed ones. The first criterion can be assayed prior to any transformation experiment by analyzing the growth rate (in our case, as a function of the increase in fresh weight) of the target cells on medium supplemented with increasing concentrations of the desired selective agents. An increasing number of selective agents (Table I118-zz) are now available, but the most commonly used products 18 L. Herrera-Estrella, M. De Block, E. Messens, J.-P. Hernalsteens, M. Van Montagu, and J. Schell, EMBO J. 2, 987 (1983). 19 C. Waldron, E. B. Murphy, J. L. Roberts, G. D. Gustafson, S. L. Armour, and S. K. Malcolm, Plant Mol. Biol. 5, 103 (1985). 2o R. M. Hauptmann, V. Vasil, P. Ozias-Akins, Z. Tabaeizadeh, S. G. Rogers, R. T. Fraley, R. B. Horsch, and I. K. Vasil, Plant Physiol. 86, 602 (1988).
[37]
431
SELECTABLE MARKERS FOR RICE TRANSFORMATION T A B L E II REPRESENTATIVE SELECTION AGENTS FOR TISSUE CULTURE
Selective agent (abbreviation) a
Detoxifying gene
Detoxification reaction
Inhibition of organeUar ribosomes Primarily inhibits 80S ribosomes Primarily inhibits 80S ribosomes
nptlI ~8
Phosphorylation
nptll j8
Phosphorylation
hpt 19
Phosphorylation
Methotrexate (Mtx)
Inhibits dihydrofolate reductase
dhrf 2°
Bleomycin (Blm)
Causes double-stranded breaks in DNA Inhibits glutamine synthase
ble 21
Mtx-resistant dihydrofolate reductase Unknown
bar 22
Acetylation
Kanamycin (Kan) G418
Hygromycin (Hyg)
Phosphinothricin (Ppt)
Mode of action
~' All chemicals are available from Sigma Chemical Company, except Ppt, which is available as Basra (Hoechst, Somerville, NJ) and contains ---200 mg/ml Ppt.
are kanamycin, hygromycin, and G418. We have evaluated the power of these as well as of phosphinothricin, bleomycin, and methotrexate to inhibit rice callus growth. Other agents that could be useful, and should be tested in similar experiments, include streptomycin, glyphosate (an herbicide that inhibits aromatic amino acid biosynthesis), and chlorsulfuron (which inhibits branched amino acid synthesis). Autoclave basic agar medium and allow it to cool to approximately 60 °. Prepare 50 mg/ml aqueous stocks of phosphinothricin, G418, kanamycin, hygromycin, and bleomycin. Filter sterilize these through 0.22-/~m disposable filters and store frozen in small aliquots (they may be kept at least 1 month). Methotrexate is dissolved in 0.1 M NaOH at a concentration of 10 mg/ml and immediately diluted into agar. Dilute each of the other chemicals into cooled medium, add 2,4-D (10 mg/ml in dimethyl sulfoxide; may be stored 6 months at - 2 0 °) to 2/~g/ml, and pour approximately 30 ml into sterile petri dishes. Using an alcohol-sterilized forceps, transfer approximately 70-100 mg of callus (we routinely use 15 individually grown callus lines) onto each dilution of every chemical tested. The growth rates of independently induced calli may differ by a factor of two to three, 21 j. Hille, F. Verheggen, P. Roelvink, H. Franssen, A. van Kammen, and P. Zabel, Plant Mol. Biol. 7, 171 (1986). 22 M. De Block, J. Botterman, M. Vandewiele, J. Dockx, C. Thoen, V. Gossel6, R. Movva, C. Thompson, M. Van Montagu, and J. Leemans, E M B O J. 6, 2513 (1987).
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REPORTERGENES
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even on nonselective medium. Thus, it is best to place a portion of each individual callus line on each medium to be compared, including one on permissive medium. Weigh calli after 5-7 days, and at 10 - 2-day intervals. This may be done by transferring material to sterile, disposable, preweighted petri dishes. Each of the subpopulations derived from a callus lineage may be weighed independently. Cover and weigh the dish to determine the new fresh weight. The calli can then be transferred onto freshly prepared plates with the corresponding concentration of selective agent. To simplify weighing, calli may also be placed onto sterile filter paper laid fiat onto the solid medium so they can be weighed en masse. If calli are maintained on filter paper, use two forceps to lift the paper into the empty petri dish. As seen in Fig. 1,z3 methotrexate (2 txg/ml), phosphinothricin (10 tzg/ ml), and hygromycin (25/xg/ml) inhibited growth within 2 weeks. G418 (-<50/xg/ml) and bleomycin (20/~g/ml) required 3 weeks to do the same. On the other hand, kanamycin inhibited rice callus growth only partially, even at concentrations 5- to 10-fold above those used to select transgenic tissue from many other plants. However, when smaller calli were used (<500 txm), lower concentrations of kanamycin (300/xg/ml) were capable of retarding growth efficiently, and protoplast division could be completely arrested at 200/xg of kanamycin/ml. This indicates that, when using kanamycin, it is important to start the selection for transformed cells soon (less than 2 weeks) after introduction of DNA into protoplasts. It should be noted that several additives including 10 mM glutamic acid, 25 mM proline, and 10 mM arginine reduce the inhibition of callus growth by 20/zg of phosphinothricin/ml from 91% to 69, 51, and 35%, respectively. It is therefore necessary to omit them, or other amino acid sources such as casein hydrolysate or coconut water, from the medium when stringent selection is desired. In our experience (Table I|I24), omitting these supplements does not markedly decrease callus formation from protoplasts. Evaluating Effectiveness of Selectable Markers
The effectiveness with which a selectable marker protects transformants depends on at least the three following factors: first, the ability of the promoter and 3' end sequences to provide adequate amounts of translatable RNA in those cells most susceptible to the selective agent 23 R. Dekeyser, B. Claes, M. Marichal, M. Van Montagu, and A. Caplan, Plant. Physiol. 90, 217 (1989). 24 j. A. Thompson, R. Abdullah, and E. C. Cocking, Plant Sci. 47, 123 (1986).
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433
"/,weight increase 600, S00, 400 300, 200 100 , - -
0 10 20 50 phosphinothricin
0 25 50 75 100 G418
0 25 .50 mg/l hygromycin
"/,weight increase 700 600
500 400 300 200 100 0 20100
bleornycin
0
2 10 20
methotrexate
0 50100250500
mglt
kanamycin
FIG. 1. Sensitivity of rice calli to different selective agents. Ten to 15 calli were transferred to media containing different concentrations of selective agent and the initial fresh weight was determined. The calli were reweighed after 10 to 12 days (white), 20 to 24 days (vertical stripes), and 28 to 31 days (slanted stripes) of incubation. The growth rate is defined as 100 times the ratio of fresh weight at day x to the initial fresh weight. The values are the averages of three independent experiments. An asterisk means there was no further increase in weight. (Reproduced from Dekeyser et al. 23 with permission by the American Society of Plant Physiologists.)
(mRNA levels can be boosted in monocots by inclusion of an intron in the primary transcript25); second, the rate with which the foreign enzyme inactivates the toxic substrate; third, the use of culture conditions that ensure that transformants are not adversely effected by metabolites from dying, untransformed cells. The capacities of the expression system (that is, the promoter, 3' end, and intron, if desired) can be assessed rapidly by assaying enzyme activity 25 j. Callis, M. Fromm, and V. Walbot, G e n e s D e v . 1, 1183 (1987).
434
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TABLE III NUMBER OF PROTOPLASTS REGENERATING TO CALLI a Medium b Treatment
KpR
KpR -
cw -
ca
K p R + Kan20 o
KpR - cw -
c a + Pptl0
Unelectroporated Electroporated Without DNA With pLD1 With pGSFR280
3.0 × 10 -3
3.0 × 10 -3
0
0
1.8 × l 0 -3 1.8 x l 0 -3 --
--1.6 x 10 -3
0 3.0 x l 0 -s --
0 0 0.7 × 10 -5
" The frequencies are the average of two independent experiments. (Reproduced from Dekeyser et al. 23 w i t h p e r m i s s i o n b y the A m e r i c a n S o c i e t y o f P l a n t P h y s i o l o g i s t s . ) b K p R , P r o t o p l a s t c u l t u r e m e d i u m c o n t a i n i n g c o c o n u t w a t e r a n d c a s a m i n o acids24; - c w , w i t h o u t c o c o n u t w a t e r ; - ca, w i t h o u t c a s a m i n o a c i d s ; + Kan200, s u p p l e m e n t e d w i t h 200 m g k a n a m y c i n / liter; + PPtlo, s u p p l e m e n t e d w i t h 10 m g p h o s p h i n o t h r i c i n / l i t e r .
produced by different constructs transiently expressed in protoplasts. The second point can be functionally investigated by comparing the growth rates of transformed and untransformed lines on selective media. The last factor generally requires frequent refreshment of the selection medium, and elimination of any plant material that does not appear healthy. Protoplast Preparation, Electroporation, and Culture Following the method of Thompson et al., 24 we isolated protoplasts from established cell suspension cultures at the third to fifth day after subculture. To enrich for small calli, the cultures are first sieved through a 250-/xm nylon mesh. One gram of drained calli is mixed with 20 ml of CPW13M medium containing 1% (w/v) cellulase RS (Yakult Honsha Co., Ltd., Nishinomiya, Japan), and 0.1% (w/v) pectolyase Y23 (Seishin Pharmaceutical Co., Ltd., Nihonbashii, Japan). The calli are incubated at room temperature in the dark on a rotary platform shaker (40 rpm) for 3 hr (until protoplasts begin to appear), followed by a further incubation at 25 ° for 2-3 hr without shaking. The digested mixture is then passed through a series of sterilized nylon sieves of 64-, 45-, and 30-/zm diameter mesh to separate undigested cell clumps from protoplasts. The protoplasts are pelleted by centrifugation at 80 g for 5 min, and washed once in CPWl3M and twice in protoplast electroporation buffer. At this point, protoplasts are pooled, counted using a hemacytometer, and adjusted to a final concentration of 5 to 7.5 × 106/ml. Two hundred microliters of this suspension is pipetted into disposable spectrophotometer cuvettes and mixed with 10/~g pLD1 z3 plasmid DNA (or an equimolar equivalent of the other plasmids), and 11 ~1 of a 3 M
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SELECTABLE MARKERS FOR RICE TRANSFORMATION
435
NaC1 stock. After l0 min on ice, an electrical shock of 375 V/cm from a 200-/.LF capacitor (the pulse time is approximately 54 msec) is delivered, and the protoplasts are left on ice for 15 min. For transient gene expression, protoplasts are gently dispersed into 5 ml KpR liquid and cultured for 48 hr in dim light at 25°. In most successful experiments (as seen in Table III) the electrical shock reduces cell viability by no more than 40-50%. The number of viable protoplasts may be estimated by diluting them into KpR medium containing 5/xg/ml fluorescein diacetate (from a stock of 500/xg/ml acetone). Viable cells fluoresce when viewed at 450-490 nm with a fluorescent light microscope through a 520nm cutoff filter. To select transformants resuspend protoplasts in 3 ml KpR liquid, heat to 45 ° (5 min), and immediately chill 10 sec on ice. Then spin 4 rain, 80 g and resuspend protoplasts to a density of approximately 3-5 × 105 viable protoplasts/ml with KpR medium containing 1% Seaplaque agarose. Quickly pipette 3.5 ml into sterile, 5-cm petri dishes, and allow medium to solidify. Cultivate the protoplasts in the dark at 25°. After 1 week, transfer each of four quarters of the agarose disks to separate 9-cm petri dishes containing 5 ml KpR medium with or without selective agent. Refresh the medium each week. Microcalli may be isolated and transferred to LS medium after 3 weeks.
Electroporation of Leaf Bases We have developed an alternative method to analyze transient gene expression in intact rice tissue instead of in protoplasts. 26 To use this technique, dehusked rice seeds are sterilized by soaking for 45 min in a solution of 0.4% (v/v) commercial bleach, 0.1% (w/v) NazCO3, 3% (w/v) NaC1, 0.15% (w/v) NaOH, and 0.01% (w/v) sodium dodecyl sulfate. They are rinsed five times with sterile water and germinated in the dark at 25° on a medium with MS salts, 1% (w/v) sucrose, and 0.8% (w/v) agar (pH 5.6). Seven days later (when the etiolated seedlings are 4 to 6 cm high) the lower leaf segment, commonly called the leaf base region, is isolated. The seedling is transferred to a large, disposable petri dish and cut with a clean, sterilized scalpel blade immediately above the site where the secondary roots pierce through the leaves. After removing the coleoptile, a second transverse cut is made approximately 3 mm above the first one. These leaf bases are then subdivided into segments with a length of 1 to 2 mm, and incubated for 3 hr in electroporation buffer (EPR) containing 0.2 mM spermidine. Next, the EPR medium is removed, the explants are washed 26 R. A. D e k e y s e r , B. Claes, R. M. U. De Rycke, M. E. Habets, M. Van Montagu, and A. B. Caplan, Plant Cell 2, 591 (1990).
436
REPORTER GENES
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twice with EPR, and the segments from 25 leaf bases (about 60 pieces in total) are transferred to a disposable spectrophotometer cuvette containing 0.2 ml EPR supplemented with 0.2 mM spermidine. Twenty micrograms of supercoiled plasmid DNA is then added to the buffer. After incubation of the DNA/explant mixture for 1 hr at room temperature (with regular, gentle shaking), add 11/zl of a 3 M NaCI stock, mix, and put the cuvette on ice for 10 min. Parallel stainless steel electrodes, 2 mm thick with inner surfaces 6 mm apart, are inserted in such a way that the leaf bases are gathered between them. Then one pulse with an electrical field strength of 375 V/cm is discharged from a 900-/zF capacitor. The homemade electroporation unit consists of an ISCO power supply connected to an array of capacitors arranged in a circuit as described. 27The explants are left on ice for 15 min and washed by three successive additions and removals of 0.3 ml KpR medium. Then the explants contained in the liquid medium are pipetted into 4.5 ml of liquid KpR medium with 2 mg 2,4-D/liter. The 5-cm diameter petri dishes are then incubated in the dark at 25°. Experiments have demonstrated that using a chloride-free EPR buffer9 containing 75 mM aspartic acid, 75 mM glutamic acid, and 4 mM calcium gluconate instead of 150 mM NaCI and 4 mM CaCI 2 increases the efficiency of transient expression approximately twofold.
Analyzing Gene Expression in Protoplasts or Cells A number of reporter systems are available, varying in convenience and sensitivity. Most of our work is first done using the neomycin phosphotransferase II (nptlI) gene. No plant species investigated to date has been found to have a phosphotransferase activity comigrating with NPTII, so that even weak expression systems can be monitored reliably.
NPTII Assay Preparation of Samples from Transformed Tissues. For assaying callus or plants, transfer approximately 0.1 to 0.2 g of tissue to a 1.6-ml Eppendorf centrifuge tube. Crush tissue in 0.1 to 0.2 ml of 2 x extraction buffer using a metal or plastic rod. The tissue is generally homogenized within 2-3 min. At that time, spin 3 min at 4°, ->9000 g, to sediment particulate debris and pipette supernatant to second tube. Chill on ice until needed. 2g To assay leaf bases, the liquid medium is carefully removed and all leaf bases are crushed within one Eppendorf tube together with 0.15 ml 2 × extraction buffer. To assay protoplasts, the medium containing the 27 M. Fromm, L. P. Taylor, and V. Walbot, Proc. Natl. Acad. Sci. U.S.A. 82, 5824 (1985). 28 B. Reiss, R. Sprengel, H. Will, and H. Schaller, Gene 30, 211 (1984).
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SELECTABLE MARKERS FOR RICE TRANSFORMATION
437
cells is gently pipetted into a 10-ml centrifuge tube, and centrifuged for 3 min at 80 g. This pellets the protoplasts at the bottom of the tube. The overlaying KpR medium is slowly removed with a capillary tube connected to a peristaltic pump, leaving behind 75 to I00/.d. Next, the protoplasts are gently resuspended by moving the tube between thumb and fingers, and transferred to an Eppendorf tube by using a wide-mouthed 200- or 1000-~1 tip pipettor. After addition of 0.25 vol (about 30 /~1) of a 4 x extraction buffer, the protoplasts are immediately chilled on ice. The protoplasts are broken by sonication for 5 to 6 sec. The sonicator probe should be set just below the surface of the liquid, and the amplitude set at 10 to 15/zm from peak to peak. To remove the cell debris, centrifuge the Eppendorf tubes at 4° for 1 min at ->9000 g in an Eppendorf centrifuge and withdraw the supernatant (containing the NPTII protein) to a precooled Eppendorf tube; store on ice. To compare the NPTII activity in different samples, equal amounts of cellular proteins are used in the NPTII assay. The protein content may be measured by the Bio-Rad (Richmond, CA) protein assay. Five microliters of each sample is mixed with 800/xl distilled water and 200/xl Bio-Rad dye, and transferred to a spectrophotometer cuvette. Before measuring the absorbance at 595 nm the reaction is allowed to proceed for 5 to I0 min. Absorption of the sample should be compared to a blank containing 5/xl of extraction buffer. If desired, a standard curve can be obtained using different amounts of bovine serum albumin (1 to 20/xg). For each NPTII determination, transfer a similar amount of protein to a precooled Eppendorf tube (we routinely load 50/zg of protein), and mix with 0.1 vol of 10 x loading buffer before loading on the nondenaturing protein gel. Preparation of Nondenaturing Gel. To prepare the protein gels, glass plates (15 x 17 cm) are thoroughly cleaned with detergent, then rinsed successively with tap water and ethanol, and dried (avoid contact between plates and skin). The plates are assembled with appropriate spacers (thickness, 1 mm) and clamps and, if necessary, sealed at the outside with a 1% (w/v) agarose solution. Next, the separation gel is poured between the glass plates up to 5 cm from their top. After carefully covering the gel solution with 0.5 cm of distilled water, the gel is allowed to polymerize (approximately 30 min at room temperature). Then the water is removed and the stacking solution is poured between the glass plates. A comb (thickness, 1 mm) with wells that can contain 100/xl of sample is inserted (avoid trapping air bubbles under the comb) and the gel is allowed to polymerize. After gently removing the comb and the bottom spacer, the gel is placed in an electrophoresis tank filled with running buffer and cooled for I hr before loading. The samples are loaded into the wells and run at 4-10 ° at 15 V/cm until the bromphenol blue marker has migrated 10 cm
438
REPORTER GENES
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into the separation gel (approximately 5 hr). Then the two glass plates are laid horizontally and gently separated. Using a scalpel blade, the upper 10 cm of the separation gel is isolated and given three 15-min washes (with shaking) in 300 ml freshly made reaction buffer (chilled at 4°). Phosphotransferase Assay. Meanwhile, 500 mg of agarose (Sigma type II) is added to 50 ml reaction buffer, rapidly heated in a microwave oven to dissolve the agarose, and cooled to 45 ° in a water bath. Subsequently, in a disposable beaker or tube, add 10/zl [T-32p]ATP (100 ~Ci, 3000 Ci/ mmol) and 100/xl of a freshly prepared aqueous kanamycin sulfate stock (50 mg/ml) to 30 ml of the agarose and mix thoroughly. Lay the rinsed separation gel into a matching tray or box, and let it equilibrate to room temperature. Next, pour the agarose solution evenly over the separation gel. Wait for the gel to solidify and cover first with one sheet of Whatman (Clifton, N J) P8 ! paper and then with two sheets of Whatman 3MM paper (all cut to size and prewet in reaction buffer), a 5-cm stack of paper towels (cut to size), and a 1-kg weight evenly distributed over the top of the towels. After 3 to 12 hr of blotting, discard the paper towels and remove the Whatman P81 paper. Wash this four times with 200 ml of washing solution for 15 min each, at 80°. The Whatman P81 paper is then covered with thin plastic wrap, and exposed to X-ray film (XAR; Kodak, Rochester, NY) using an intensifying screen at - 70°. Exposure time varies from 10 min to 72 hr, depending on the strength of the NPTII signal. As controls for the NPTII assay, we used protein extracts from an untransformed tobacco plant (blank) and a tobacco plant transformed with the P2'-nptII construct (positive control). NPTII activity is detected by production and binding of phosphorylated kanamycin to the Whatman P81 paper. One often sees two slower migrating bands above this that are also detected in untransformed material and probably correspond to phosphorylated proteins. To quantitate the NPTII activity, the autoradiogram is matched with the Whatman P81 paper, the NPTII spots are located, cut out with a scalpel blade, air dried, submerged in scintillation liquid, and counted in a Beckman (Palo Alto, CA) scintillation counter.
Conclusions Of the six promoters tested in protoplasts, and the four in leaf bases (Table IV), the two strongest and least influenced by the source of protoplast or explant are derived from the mannopine synthase 2' gene and the cauliflower mosaic virus (CaMV) 35S transcript. It is useful to note that the ratio of activities of the P35S and Pnos constructs measured in transient expression assays 23 (approximately 3.6) is similar to the ratio of transfor-
[37]
SELECTABLE
MARKERS
439
FOR RICE TRANSFORMATION
T A B L E IV RELATIVE N P T I I ACTIVITY (%) PRODUCED BY DIFFERENT CHIMERIC GENES IN PROTOPLASTS AND LEAF BASESa Protoplasts from P r o m o t e r fused to nptlI gene (reference) N o n e (23, 26) P2' (23, 26)
P35S (23, 26) Pnos (23) PI' (23) Pz4 (23) P4.7 (23) P e x t e n s i n (26) Pcab (26)
E x p l a n t s from
Suspension cultures
Leaves
L e a f bases (L/D) b
Leaves (L/D)
ND 100 40 11 10 0.4 0.2 ---
ND 100 -8 -0.4 0.2 ---
ND/ND 93/100 95/98 ------/6 ND/ND
ND/ND 20/100 26/96 -----83/ND
" In each column, all e x p e r i m e n t s have been normalized to P 2' -dri ve n N P T I I activity. ( R e p r o d u c e d from D e k e y s e r et al. 26 with permission by the A m e r i c a n Society of Plant Physiologists.) b L e a f bases or leaves have been p r e g r o w n and maintained in light (L) or da rk (D) for 4 days after electroporation. ND, Not detected.
mation frequencies obtained with each promoter under selective conditions (2.6 and 3.0). 6 This indicates transient assays measure parameters that are useful for predicting transformation efficiencies. We have measured the growth rates of calli containing each of four different selectable markers (Pnos : nptII, P2' : nptII, P35S : bar, and P l ' : d h f r ) on different concentrations of the corresponding selective agents. These calli contained similar numbers of inserts, 23 and as seen in Table III were isolated at comparable frequencies, although some were selected on 200/xg kanamycin/ml, and some with 10/xg phosphinothricin/ ml. Similarly, there were comparable levels of NPTII activity in the calli analyzed, irrespective of how they were initially selected. 23 The first relevant observation made on the regenerate material was that there was no significant difference in the growth rates of transformed and untransformed calli on 0, 100, or 500/zg kanamycin/ml. On the other hand, the same transformed lines grew 8-10 times faster than untransformed calli on both 100 and 500 /xg G418/ml, for which rice has no endogenous resistance. Thus, to minimize the frequency of escapes from selection, one should use G418 instead of kanamycin whenever possible. Calli containing the P35S : bar construct also grew well under selection, in fact, 10-15 times faster than untransformed calli on 10 or 50 /xg of
440
REPORTER GENES
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phosphinothricin/ml. Calli containing PI' : dhfr did not grow rapidly on either 2 or I0/~g methotrexate/ml. All untransformed material died within 1 week. Either of these selection systems could, therefore, be used in place of the aminoglycoside detoxification system. However, it must be noted that the majority of fertile, transgenic plants obtained to date have been isolated using P35S : h p t 3'5'6'29
Variables in Gene Expression in Transformed Material The expression of transferred genes often varies from transformant to transformant. Sometimes the new genes are poorly expressed in some lines, or even silent. 29'3° In other cases, apparently homogeneous calli do not express fl-glucuronidase activity uniformly,3° or NPTII-deficient plants regenerate from NPTII-positive material, z A priori, these phenomena might indicate that the originally selected colonies were in fact polyclonal; however, other factors beyond the control of the investigator might produce similar results. For example, gene expression can be reduced by changes in DNA methylation or organization occurring in response to tissue culture conditions. 3L32 In other cases, activation or inactivation of nearby chromosomal promoters could silence gene transcription or generate antisense transcripts to reduce the expression of the foreign gene. On the other hand, foreign gene expression in most calli stabilizes within 3 to 5 months of culture, 8 and can remain constant in the absence of selection.Z,3,5,6, 30
Although the presence of the selectable marker can sometimes be verified by assaying for the corresponding enzyme activity, the presence of the cotransferred genes that have no overt phenotype can be ascertained only by performing Southern hybridization. 33 To answer questions about the organization of the new genes, one should always use restriction enzymes with targets that are generally not methylated in eukaryotes such as BgllI, EcoRI, or HindlII. Preferably, at least one (or a mixture of two) of the enzymes should be chosen because it cuts twice within the gene under investivation and generates a fragment that must be intact for the gene to function. In appropriate reconstruction experiments, one can compare the intensity of hybridization of probes to genomic DNA and to 29 R. Terada and K. Shimamoto, Mol. Gen. Genet. 220, 389 (1990). 3o W. Zhang and R. Wu, Theor. Appl. Genet. 76, 835 (1988). 31 p. T. H. Brown, J. Kyozuka, Y. Sukekiyo, Y. Kimura, K. Shimamoto, and H. L6rz, Mol. Gen. Genet. 223, 324 (1990). 32 E. MOiler, P. T. H. Brown, S. Hartke, and H. LOrz, Theor. Appl. Genet. 80, 673 (1990). 33 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980.
[38]
THAUMATIN II
441
similarly cut plasmid to estimate the copy number of the foreign gene. A single-copy equivalent of a 10-kb sequence in rice, assuming a diploid genome size of 4 x 108 bp, is approximately 2.5 × 10 -5/xg//zg genomic DNA. Similar analyses with a second restriction enzyme that cuts once, or not at all, in the transferred D N A can clarify the number of independent integration sites of the gene in the host sequence. We have analyzed seven transformed rice callus lines using BgllI, EcoRI, and a mixture of EcoRV and BstEII. Forty percent had single copies of the selected markers. Neither these transformants, nor others listed in Table IV, showed a consistent correlation between copy number and either gene expression or levels of resistance. Because deletions and mutations may be c o m m o n during integration of foreign sequences, it is possible many of these genes are not expressed. Acknowledgments This work was supported by grants from the Rockefeller Foundation (RF 86058 #59) and the Services of the Prime Minister (U.I.A.P. #120CO187). R.D. was a Research Assistant of the National Fund for Scientific Research (Belgium).
[38] T h a u m a t i n
II: A S w e e t M a r k e r
G e n e f o r U s e in P l a n t s
By MICHAEL WITTY Introduction Thaumatin II is a natural product of the West African plant Thaumatococcus daniellii. The fruit of this plant produces a family of five or more extremely sweet proteins ~called thaumatins that are traditionally used by West Africans to sweeten food and beverages. 2 In 1839 DanieU, the first European to write ofthaumatin, described it as having " . . . anindescribable yet intense degree of dulcidity . . . . ,,2 Thaumatin has long been a substance well known for the intensity of its taste and the small amount needed to produce an obvious sensation. Thaumatin solutions taste sweet at concentrations as low as 10 -8 M . 3 Thaumatins also lower the taste
l H. van der Wel and K. Loeve, Eur. J. Biochem. 31, 221 (1972). 2 W. F. Daniell, Pharm. J. 14, 158 (1855). 3 j. D. Higginbotham, in " D e v e l o p m e n t s in Sweeteners I , " p. 87. Applied Science Publ., London, 1979.
METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
REPORTER GENES
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9. Transblot for 3-4 hr at 40 V; make sure that the nitrocellulose is toward the anode. 10. Incubate the filter for 2-4hr at room temperature or overnight at 4° in PBS-ovalbumin buffer [150 mM NaCI, 12.6 mM NaHzPO 4 • 2H20, 14 mM K H z P O 4, pH 7.4, 0.5% (w/v) ovalbumin] with rocking. 11. Soak the filter at room temperature for 2 hr with gentle shaking in anti-PAT antibody solution. 12. Wash three times in PBS containing 0.1% (v/v) Triton X-100 for 10 min. 13. Incubate for 2 hr at room temperature in a second antibody solution (alkaline phosphatase-conjugated anti-rabbit IgG; Sigma). 14. Wash once with PBS [with 0.1% (v/v) Triton X-100] for 30 min and three times with PBS for 30 min. 15. Add freshly made staining solution [30 mg p-nitro blue tetrazolium chloride (NBT; Bio-Rad) in 1 ml 70% (v/v) N,N-dimethylformamide, 15 mg 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP; Sigma) in 1 ml 100% N,N-dimethylformamide, 98 ml carbonate buffer (0.1 M NaHCO 3 + 1.0 mM MgCI2) (pH 9.8)] and shake until the reaction product is visible. 16. Rinse the filter in distilled water and dry between two sheets of Whatman 3MM paper in the dark.
[37] Selectable Markers for R i c e T r a n s f o r m a t i o n By
A L L A N C A P L A N , R U D Y DEKEYSER,
and MARC V A N MONTAGU
Introduction
Choosing Convenient Method to Introduce D N A A number of techniques have been developed to transfer DNA into rice. Some techniques, such as polyethylene glycol (PEG)-mediated or electroporation-mediated gene transfer, have been used repeatedly by several groups, 1-9 and are becoming well-established routines for genetic I K. Toriyama, Y. Arimoto, H. Uchimiya, and K. Hinata, Bio/Technology 6, 1072 (1988). 2 H. M. Zhang, H. Yang, E. L. Rech, T. J. Golds, A. S. Davis, B. J. Mulligan, E. C. Cocking, and M. R. Davey, Plant Cell Rep. 7, 379 (1988). 3 K. Shimamoto, R. Terada, T. Izawa, and H. Fujimoto, Nature (London) 338, 274 (1989). 4 R. Matsuki, H. Onodera, T. Yamauchi, and H. Uchimiya, Mol. Gen. Genet. 220, 12 (1989). 5 S. K. Datta, A. Peterhans, K. Datta, and I. Potrykus, Bio/Technology 8, 736 (1990).
METHODS IN ENZYMOLOGY,VOL. 216
Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[37]
427
SELECTABLE MARKERS FOR RICE TRANSFORMATION
TABLE I REPRESENTATIVE TRANSFORMATION EFF1CIENCIES OF RICE PROTOPLASTS DNA Selectable marker~
concentration (/xg/ml)
P35S:nptlI P35S:nptlI P35S:hpt P35S:hpt P35S:hpt Pnos:hpt P35S:hpt P35S:nptlI P35S:nptlI P35S:hpt
16 20 10 1 1.3 40 40 50 75 20
Transformation frequency (10 -6)
Estimated
introduction b
Selective agent (/zg/ml) c
copy number
Ref.
E E E E P P P P P E
G418 (20) Kan (100) Hyg (20) Hyg (50) Hyg (25) Hyg (50, 100) Hyg (50, 100) Kan (100) Kan ( 2 0 0 ) Hyg (50)
4 280 Up to 6500 120-200 5-44 d 76; 59 197; 176 20 3250-6500 d 134-137
1-2 1-2 2-10 ->3 50-100 -1-10 2-10 20-30 1-10
1 2 3 4 5 6 6 7 8 9
Method of
'~ P35S, cauliflower mosaic virus (CaMV) promoter; Pnos, nopaline synthase promoter; nptlI, neomycin phosphotransferase II gene; hpt, hygromycin phosphotransferase gene. b E, Electroporation; P, PEG. " Kan, Kanamycin; Hyg, hygromycin. d Indica variety of rice; all other experiments use japonica varieties.
engineering projects. Others including pollen tube transformation, ~0particle guns, 1~and Agrobacterium tumefaciens, 12which have been introduced more recently, involve more specialized skills, and hence seem more difficult to apply repeatedly. Genes transferred by any of the first four methods mentioned rely on host D N A repair systems to link the foreign genes to the chromosomes. The integration sites may be distributed at random along the sequences of the introduced DNA, and thus may disrupt the gene being transferred. However, Agrobacterium and its tumor-inducing (Ti) plasmid-mediated system of cell transformation should be able to transfer D N A in a defined manner producing predictable end points for integration. Electroporation and PEG-mediated gene transfer do not differ greatly either in terms of efficiency or ease of application (Table I). Each functions 6 A. Hayashimoto, Z. Li, and N. Murai, Plant Physiol. 93, 857 (1990). 7 A. Peterhans, S. K. Datta, K. Datta, G. J. Goodall, I. Potrykus, and J. Paszkowski, Mol. Gen. Genet. 222, 361 (1990). 8 j. Peng, L. J. Lyznik, L. Lee, and T. K. Hodges, Plant Cell Rep. 9, 168 (1990). 9 y . Tada, M. Sakamoto, and T. Fujimura, Theor. Appl. Genet, 80, 475 (1990). z0 Z.-X. Luo and R. Wu, Plant Mol. Biol. Rep. 6, 165 (1988). tl M. E. Fromm, F. Morrish, C. Armstrong, R. Williams, J. Thomas, and T. M. Klein, Bio/ Technology 8, 833 (1990). 12 D. M. Raineri, P. Bottino, M. P. Gordon, and E. W. Nester, Bio/Technology 8, 33 (1990).
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REPORTER GENES
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by opening pores through which DNA can diffuse into the cell, and eventually into the nucleus, where transcription is possible. Both of these methods require similar amounts of purified DNA (generally ranging from 10 to 40 ~g/sample). For the sake of convenience, it is advisable to maintain the genes that are to be transferred on high-copy number, stably segregating Escherichia coli vectors such as those derived from the modified ColE1 origin present in plasmids such as pUC18. ~3 Whereas it has been demonstrated that plasmids as large as 200 kilobases (kb) can be electroporated into protoplasts, 14it is prudent to keep in mind that larger plasmids often yield less DNA during isolation from bacteria. One way of circumventing size constraints is to separate the sequences to be transferred into two sets. For example, the selected marker and the gene under investigation may be cloned on individual vectors and mixed prior to DNA transfer. Molar ratios of 1 : 2, 1 : 1, or 2 : 1 (selected-unselected) yield similar frequencies of cotransformation that differ as much between replicates as between planned variations ( 1 4 - 8 5 % ) . 4,7,9 For some experiments such two-component systems may offer several advantages. First, the cotransformed vector may contain the same restriction sites or expression sequences present in the selected marker without complicating the cloning strategy or increasing the risk of homology-dependent deletion formation that can occur between repeated sequences in the same bacteria. Second, the selected marker and the nonselected gene can integrate at independent loci and thus segregate during meiosis. In this way, primary transformants can produce descendants that are no longer drug resistant, yet retain the unselected gene of interest. Such plants thus have very little extraneous DNA, and can be retransformed with the same selectable marker, should later experiments require the introduction of other genes.
Materials
Protoplast and Plant Culture Media and Solutions Linsmaier and Skoog (LS) medium15:1 liter of deionized water, 4.4 g of Murashige and Skoog salts (Sigma Chemical Co., St. Louis, MO), 1 mg of thiamin hydrochloride, 100 mg of myo-inositol, 30 g of sucrose, 4 g of agarose, pH 5.7. Autoclave, cool to 55°, and add 2 mg of 2,4dichlorophenoxyacetic acid (2,4-D) dissolved in dimethyl sulfoxide (DMSO; from a stock of 10 mg/ml) 13 C. Yanisch-Perron, J. Vieira, and J. Messing, Gene 33, 103 (1985). 14 W° H. R. Langridge, B. J. Li, and A. A. Szalay, Plant Cell Rep. 4, 355 (1985). 15 E. M. Linsrnaier and F. Skoog, Physiol. Plant. 18, 100 (1965).
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SELECTABLE MARKERS FOR RICE TRANSFORMATION
429
CPW13M16:1 liter of deionized water, 101 mg of KNO3, 27.2 mg of KHzPO 4, 1480 mg of CaC12- 2H20, 0.025 mg of MgSO4" 7H20, 0.16 mg of KI, 0.025 mg of CuSO4.5H20, 975 mg of 2-(N-morpholino) ethanesulfonic acid, 130 g of mannitol, pH 5.8. Autoclave KpR (per liter of deionized H20)17: As this medium is quite complex, it is advisable to make several stock solutions. Macronutrients can be added as dry powder: 1900 mg of KNO 3, 600 mg of NH4NO 3, 600 mg of CaCI z • 2 H 2 0 , 300 mg of M g S O 4 • 7 H 2 0 , 300 mg of KCI, 170 mg of KHzPO 4. Micronutrients are added as 10 ml of a I00 x stock solution containing (per liter) 1000 mg of MnSO 4 • HzO, 300 mg of H3BO3,200 mg of Z n S O 4 • 7 H 2 0 , 75 mg of KI, 25 mg of N a 2 M o O 4 • 2H20, 2.5 mg o f C u S O 4 • 5 H 2 0 , and 2.5 mg of CoC1z • 6H20. Organic acids are added as 10 ml of a I00 x stock solution containing (per liter) 1 g of citric acid, 1 g of malic acid, 1 g of fumaric acid, and 0.5 g of sodium pyruvate. Adjust pH of stock solution to 5.5 with NH4OH. Several vitamins are added as 10 ml of a 100 x stock containing (per liter) 100 mg of nicotinamide, 100 mg of ascorbic acid, 100 mg of pyridoxinhydrochloride, 100 mg of thiamin hydrochloride, 50 mg of calcium pantothenate, 50 mg of choline chloride, 20 mg of folic acid, and 10 mg of riboflavin. KpR medium is completed by addition of 100 mg of m y o inositol (as dry powder), 10/~1 ofp-aminobenzoic acid (1 mg/ml) stock in H 2 0 ) , 10 /zl of vitamin A (0.5 mg/ml stock in ethanol), 10/zl of vitamin D3 (0.5 mg/ml stock in ethanol), 10/zl of vitamin B12 (1 mg/ ml stock in ethanol), and 1.0 ml of biotin (0.5 mg/ml stock in water). Add 1.25 g of from a dry mix consisting of D-sucrose (10 g), D-fructose (5 g), D-ribose (5 g), D-xylose (5 g), D-mannose (5 g), D-cellobiose (5 g), L-rhamnose (5 g), D-mannitol (5 g), D-sorbitol (5 g). Iron is supplied to the medium as FeSO4" 7H20 (28.75 mg) and Na 2 EDTA (37.25 mg). In our experiments we use 10% (w/v) glucose as osmoticum, omit the casamino acids and the coconut water, and use 0.5 mg of 2,4-D (10 mg/ml stock in DMSO), 1 mg of naphthaleneacetic acid (10 mg/ml stock in DMSO), and 0.5 mg of zeatin (5 mg/ml stock in DMSO) as hormones. The final pH of the solution should be 5.7. In general, the medium is filter sterilized using a 0.2-~m filter unit (Nalgene, Nalge, Rochester, NY), and stored at 4 °. To make agarose for embedding protoplasts, use deionized water to prepare a 2% Seaplaque agarose (FMC, Rockland, ME) solution, autoclave, cool to 45 °, and mix with an equal volume of 2 × KpR solution prior to use 16 E. M. Frearson, J. B. Power, and E. C. Cocking, Dev. Biol. 33, 130 (1973). 17 K. N. Kao, Mol. Gen. Genet. 150, 225 (1977).
430
REPORTER GENES
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Electroporation buffer (EPR): 10 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), pH 7.2, 4 mM CaCI2, 10% (w/v) glucose
Solutions for NPTII Assay Use distilled water for all solutions and buffers. Extraction buffer (4 × ): 100 mM Tris-HC1 (pH 6.8), 0.3 mg/ml leupeptin, 2% (v/v) 2-mercaptoethanol Loading buffer (10 × ): 50% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol, 0.5% (v/v) sodium dodecyl sulfate (SDS), 0.005% (v/v) bromphenol blue Separation gel: For 30 ml, 10 ml of 30% (w/v) acrylamide, 3.9 ml of 2% (w/v) bisacrylamide, 11.2 ml of 1 M Tris-HCl (pH 8.7), 4.5 ml of water. When ready to pour gel, add 0.2 ml of 10% (w/v) ammonium persulfate (APS) and 0.04 ml of N,N,N',N'-tetramethylethylenediamine) (TEMED) Stacking gel: For 10 ml, 1.67 ml of 30% (w/v) acrylamide, 1.3 ml of 2% (w/v) bisacrylamide, 1.25 ml of 100 M Tris-HC1 (pH 6.8), 5.6 ml of water. When ready to pour, add 0.1 ml of 10% (w/v) APS and 0.01 ml of TEMED Running buffer: 6 g/liter Tris, 14.9 g/liter glycine (do not adjust pH) Reaction buffer: 50 mM Tris-HC1 (pH 7.5), 200 mM NH4C1, 25 mM MgC12, 0.5 mM dithiothreitol (DTT) Washing buffer: 100 mM Na2HPO 4 (pH 7.0; adjusted with HC1) Methods and Discussion
Possible Selective Agents The efficiency of any selection technique is determined both by the ability of the selective agent to inhibit untransformed cells, and by the ability of the detoxifying gene to protect transformed ones. The first criterion can be assayed prior to any transformation experiment by analyzing the growth rate (in our case, as a function of the increase in fresh weight) of the target cells on medium supplemented with increasing concentrations of the desired selective agents. An increasing number of selective agents (Table I118-zz) are now available, but the most commonly used products 18 L. Herrera-Estrella, M. De Block, E. Messens, J.-P. Hernalsteens, M. Van Montagu, and J. Schell, EMBO J. 2, 987 (1983). 19 C. Waldron, E. B. Murphy, J. L. Roberts, G. D. Gustafson, S. L. Armour, and S. K. Malcolm, Plant Mol. Biol. 5, 103 (1985). 2o R. M. Hauptmann, V. Vasil, P. Ozias-Akins, Z. Tabaeizadeh, S. G. Rogers, R. T. Fraley, R. B. Horsch, and I. K. Vasil, Plant Physiol. 86, 602 (1988).
[37]
431
SELECTABLE MARKERS FOR RICE TRANSFORMATION T A B L E II REPRESENTATIVE SELECTION AGENTS FOR TISSUE CULTURE
Selective agent (abbreviation) a
Detoxifying gene
Detoxification reaction
Inhibition of organeUar ribosomes Primarily inhibits 80S ribosomes Primarily inhibits 80S ribosomes
nptlI ~8
Phosphorylation
nptll j8
Phosphorylation
hpt 19
Phosphorylation
Methotrexate (Mtx)
Inhibits dihydrofolate reductase
dhrf 2°
Bleomycin (Blm)
Causes double-stranded breaks in DNA Inhibits glutamine synthase
ble 21
Mtx-resistant dihydrofolate reductase Unknown
bar 22
Acetylation
Kanamycin (Kan) G418
Hygromycin (Hyg)
Phosphinothricin (Ppt)
Mode of action
~' All chemicals are available from Sigma Chemical Company, except Ppt, which is available as Basra (Hoechst, Somerville, NJ) and contains ---200 mg/ml Ppt.
are kanamycin, hygromycin, and G418. We have evaluated the power of these as well as of phosphinothricin, bleomycin, and methotrexate to inhibit rice callus growth. Other agents that could be useful, and should be tested in similar experiments, include streptomycin, glyphosate (an herbicide that inhibits aromatic amino acid biosynthesis), and chlorsulfuron (which inhibits branched amino acid synthesis). Autoclave basic agar medium and allow it to cool to approximately 60 °. Prepare 50 mg/ml aqueous stocks of phosphinothricin, G418, kanamycin, hygromycin, and bleomycin. Filter sterilize these through 0.22-/~m disposable filters and store frozen in small aliquots (they may be kept at least 1 month). Methotrexate is dissolved in 0.1 M NaOH at a concentration of 10 mg/ml and immediately diluted into agar. Dilute each of the other chemicals into cooled medium, add 2,4-D (10 mg/ml in dimethyl sulfoxide; may be stored 6 months at - 2 0 °) to 2/~g/ml, and pour approximately 30 ml into sterile petri dishes. Using an alcohol-sterilized forceps, transfer approximately 70-100 mg of callus (we routinely use 15 individually grown callus lines) onto each dilution of every chemical tested. The growth rates of independently induced calli may differ by a factor of two to three, 21 j. Hille, F. Verheggen, P. Roelvink, H. Franssen, A. van Kammen, and P. Zabel, Plant Mol. Biol. 7, 171 (1986). 22 M. De Block, J. Botterman, M. Vandewiele, J. Dockx, C. Thoen, V. Gossel6, R. Movva, C. Thompson, M. Van Montagu, and J. Leemans, E M B O J. 6, 2513 (1987).
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REPORTERGENES
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even on nonselective medium. Thus, it is best to place a portion of each individual callus line on each medium to be compared, including one on permissive medium. Weigh calli after 5-7 days, and at 10 - 2-day intervals. This may be done by transferring material to sterile, disposable, preweighted petri dishes. Each of the subpopulations derived from a callus lineage may be weighed independently. Cover and weigh the dish to determine the new fresh weight. The calli can then be transferred onto freshly prepared plates with the corresponding concentration of selective agent. To simplify weighing, calli may also be placed onto sterile filter paper laid fiat onto the solid medium so they can be weighed en masse. If calli are maintained on filter paper, use two forceps to lift the paper into the empty petri dish. As seen in Fig. 1,z3 methotrexate (2 txg/ml), phosphinothricin (10 tzg/ ml), and hygromycin (25/xg/ml) inhibited growth within 2 weeks. G418 (-<50/xg/ml) and bleomycin (20/~g/ml) required 3 weeks to do the same. On the other hand, kanamycin inhibited rice callus growth only partially, even at concentrations 5- to 10-fold above those used to select transgenic tissue from many other plants. However, when smaller calli were used (<500 txm), lower concentrations of kanamycin (300/xg/ml) were capable of retarding growth efficiently, and protoplast division could be completely arrested at 200/xg of kanamycin/ml. This indicates that, when using kanamycin, it is important to start the selection for transformed cells soon (less than 2 weeks) after introduction of DNA into protoplasts. It should be noted that several additives including 10 mM glutamic acid, 25 mM proline, and 10 mM arginine reduce the inhibition of callus growth by 20/zg of phosphinothricin/ml from 91% to 69, 51, and 35%, respectively. It is therefore necessary to omit them, or other amino acid sources such as casein hydrolysate or coconut water, from the medium when stringent selection is desired. In our experience (Table I|I24), omitting these supplements does not markedly decrease callus formation from protoplasts. Evaluating Effectiveness of Selectable Markers
The effectiveness with which a selectable marker protects transformants depends on at least the three following factors: first, the ability of the promoter and 3' end sequences to provide adequate amounts of translatable RNA in those cells most susceptible to the selective agent 23 R. Dekeyser, B. Claes, M. Marichal, M. Van Montagu, and A. Caplan, Plant. Physiol. 90, 217 (1989). 24 j. A. Thompson, R. Abdullah, and E. C. Cocking, Plant Sci. 47, 123 (1986).
[37]
SELECTABLE MARKERS FOR RICE TRANSFORMATION
433
"/,weight increase 600, S00, 400 300, 200 100 , - -
0 10 20 50 phosphinothricin
0 25 50 75 100 G418
0 25 .50 mg/l hygromycin
"/,weight increase 700 600
500 400 300 200 100 0 20100
bleornycin
0
2 10 20
methotrexate
0 50100250500
mglt
kanamycin
FIG. 1. Sensitivity of rice calli to different selective agents. Ten to 15 calli were transferred to media containing different concentrations of selective agent and the initial fresh weight was determined. The calli were reweighed after 10 to 12 days (white), 20 to 24 days (vertical stripes), and 28 to 31 days (slanted stripes) of incubation. The growth rate is defined as 100 times the ratio of fresh weight at day x to the initial fresh weight. The values are the averages of three independent experiments. An asterisk means there was no further increase in weight. (Reproduced from Dekeyser et al. 23 with permission by the American Society of Plant Physiologists.)
(mRNA levels can be boosted in monocots by inclusion of an intron in the primary transcript25); second, the rate with which the foreign enzyme inactivates the toxic substrate; third, the use of culture conditions that ensure that transformants are not adversely effected by metabolites from dying, untransformed cells. The capacities of the expression system (that is, the promoter, 3' end, and intron, if desired) can be assessed rapidly by assaying enzyme activity 25 j. Callis, M. Fromm, and V. Walbot, G e n e s D e v . 1, 1183 (1987).
434
REPORTERGENES
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TABLE III NUMBER OF PROTOPLASTS REGENERATING TO CALLI a Medium b Treatment
KpR
KpR -
cw -
ca
K p R + Kan20 o
KpR - cw -
c a + Pptl0
Unelectroporated Electroporated Without DNA With pLD1 With pGSFR280
3.0 × 10 -3
3.0 × 10 -3
0
0
1.8 × l 0 -3 1.8 x l 0 -3 --
--1.6 x 10 -3
0 3.0 x l 0 -s --
0 0 0.7 × 10 -5
" The frequencies are the average of two independent experiments. (Reproduced from Dekeyser et al. 23 w i t h p e r m i s s i o n b y the A m e r i c a n S o c i e t y o f P l a n t P h y s i o l o g i s t s . ) b K p R , P r o t o p l a s t c u l t u r e m e d i u m c o n t a i n i n g c o c o n u t w a t e r a n d c a s a m i n o acids24; - c w , w i t h o u t c o c o n u t w a t e r ; - ca, w i t h o u t c a s a m i n o a c i d s ; + Kan200, s u p p l e m e n t e d w i t h 200 m g k a n a m y c i n / liter; + PPtlo, s u p p l e m e n t e d w i t h 10 m g p h o s p h i n o t h r i c i n / l i t e r .
produced by different constructs transiently expressed in protoplasts. The second point can be functionally investigated by comparing the growth rates of transformed and untransformed lines on selective media. The last factor generally requires frequent refreshment of the selection medium, and elimination of any plant material that does not appear healthy. Protoplast Preparation, Electroporation, and Culture Following the method of Thompson et al., 24 we isolated protoplasts from established cell suspension cultures at the third to fifth day after subculture. To enrich for small calli, the cultures are first sieved through a 250-/xm nylon mesh. One gram of drained calli is mixed with 20 ml of CPW13M medium containing 1% (w/v) cellulase RS (Yakult Honsha Co., Ltd., Nishinomiya, Japan), and 0.1% (w/v) pectolyase Y23 (Seishin Pharmaceutical Co., Ltd., Nihonbashii, Japan). The calli are incubated at room temperature in the dark on a rotary platform shaker (40 rpm) for 3 hr (until protoplasts begin to appear), followed by a further incubation at 25 ° for 2-3 hr without shaking. The digested mixture is then passed through a series of sterilized nylon sieves of 64-, 45-, and 30-/zm diameter mesh to separate undigested cell clumps from protoplasts. The protoplasts are pelleted by centrifugation at 80 g for 5 min, and washed once in CPWl3M and twice in protoplast electroporation buffer. At this point, protoplasts are pooled, counted using a hemacytometer, and adjusted to a final concentration of 5 to 7.5 × 106/ml. Two hundred microliters of this suspension is pipetted into disposable spectrophotometer cuvettes and mixed with 10/~g pLD1 z3 plasmid DNA (or an equimolar equivalent of the other plasmids), and 11 ~1 of a 3 M
[37]
SELECTABLE MARKERS FOR RICE TRANSFORMATION
435
NaC1 stock. After l0 min on ice, an electrical shock of 375 V/cm from a 200-/.LF capacitor (the pulse time is approximately 54 msec) is delivered, and the protoplasts are left on ice for 15 min. For transient gene expression, protoplasts are gently dispersed into 5 ml KpR liquid and cultured for 48 hr in dim light at 25°. In most successful experiments (as seen in Table III) the electrical shock reduces cell viability by no more than 40-50%. The number of viable protoplasts may be estimated by diluting them into KpR medium containing 5/xg/ml fluorescein diacetate (from a stock of 500/xg/ml acetone). Viable cells fluoresce when viewed at 450-490 nm with a fluorescent light microscope through a 520nm cutoff filter. To select transformants resuspend protoplasts in 3 ml KpR liquid, heat to 45 ° (5 min), and immediately chill 10 sec on ice. Then spin 4 rain, 80 g and resuspend protoplasts to a density of approximately 3-5 × 105 viable protoplasts/ml with KpR medium containing 1% Seaplaque agarose. Quickly pipette 3.5 ml into sterile, 5-cm petri dishes, and allow medium to solidify. Cultivate the protoplasts in the dark at 25°. After 1 week, transfer each of four quarters of the agarose disks to separate 9-cm petri dishes containing 5 ml KpR medium with or without selective agent. Refresh the medium each week. Microcalli may be isolated and transferred to LS medium after 3 weeks.
Electroporation of Leaf Bases We have developed an alternative method to analyze transient gene expression in intact rice tissue instead of in protoplasts. 26 To use this technique, dehusked rice seeds are sterilized by soaking for 45 min in a solution of 0.4% (v/v) commercial bleach, 0.1% (w/v) NazCO3, 3% (w/v) NaC1, 0.15% (w/v) NaOH, and 0.01% (w/v) sodium dodecyl sulfate. They are rinsed five times with sterile water and germinated in the dark at 25° on a medium with MS salts, 1% (w/v) sucrose, and 0.8% (w/v) agar (pH 5.6). Seven days later (when the etiolated seedlings are 4 to 6 cm high) the lower leaf segment, commonly called the leaf base region, is isolated. The seedling is transferred to a large, disposable petri dish and cut with a clean, sterilized scalpel blade immediately above the site where the secondary roots pierce through the leaves. After removing the coleoptile, a second transverse cut is made approximately 3 mm above the first one. These leaf bases are then subdivided into segments with a length of 1 to 2 mm, and incubated for 3 hr in electroporation buffer (EPR) containing 0.2 mM spermidine. Next, the EPR medium is removed, the explants are washed 26 R. A. D e k e y s e r , B. Claes, R. M. U. De Rycke, M. E. Habets, M. Van Montagu, and A. B. Caplan, Plant Cell 2, 591 (1990).
436
REPORTER GENES
[37]
twice with EPR, and the segments from 25 leaf bases (about 60 pieces in total) are transferred to a disposable spectrophotometer cuvette containing 0.2 ml EPR supplemented with 0.2 mM spermidine. Twenty micrograms of supercoiled plasmid DNA is then added to the buffer. After incubation of the DNA/explant mixture for 1 hr at room temperature (with regular, gentle shaking), add 11/zl of a 3 M NaCI stock, mix, and put the cuvette on ice for 10 min. Parallel stainless steel electrodes, 2 mm thick with inner surfaces 6 mm apart, are inserted in such a way that the leaf bases are gathered between them. Then one pulse with an electrical field strength of 375 V/cm is discharged from a 900-/zF capacitor. The homemade electroporation unit consists of an ISCO power supply connected to an array of capacitors arranged in a circuit as described. 27The explants are left on ice for 15 min and washed by three successive additions and removals of 0.3 ml KpR medium. Then the explants contained in the liquid medium are pipetted into 4.5 ml of liquid KpR medium with 2 mg 2,4-D/liter. The 5-cm diameter petri dishes are then incubated in the dark at 25°. Experiments have demonstrated that using a chloride-free EPR buffer9 containing 75 mM aspartic acid, 75 mM glutamic acid, and 4 mM calcium gluconate instead of 150 mM NaCI and 4 mM CaCI 2 increases the efficiency of transient expression approximately twofold.
Analyzing Gene Expression in Protoplasts or Cells A number of reporter systems are available, varying in convenience and sensitivity. Most of our work is first done using the neomycin phosphotransferase II (nptlI) gene. No plant species investigated to date has been found to have a phosphotransferase activity comigrating with NPTII, so that even weak expression systems can be monitored reliably.
NPTII Assay Preparation of Samples from Transformed Tissues. For assaying callus or plants, transfer approximately 0.1 to 0.2 g of tissue to a 1.6-ml Eppendorf centrifuge tube. Crush tissue in 0.1 to 0.2 ml of 2 x extraction buffer using a metal or plastic rod. The tissue is generally homogenized within 2-3 min. At that time, spin 3 min at 4°, ->9000 g, to sediment particulate debris and pipette supernatant to second tube. Chill on ice until needed. 2g To assay leaf bases, the liquid medium is carefully removed and all leaf bases are crushed within one Eppendorf tube together with 0.15 ml 2 × extraction buffer. To assay protoplasts, the medium containing the 27 M. Fromm, L. P. Taylor, and V. Walbot, Proc. Natl. Acad. Sci. U.S.A. 82, 5824 (1985). 28 B. Reiss, R. Sprengel, H. Will, and H. Schaller, Gene 30, 211 (1984).
[37]
SELECTABLE MARKERS FOR RICE TRANSFORMATION
437
cells is gently pipetted into a 10-ml centrifuge tube, and centrifuged for 3 min at 80 g. This pellets the protoplasts at the bottom of the tube. The overlaying KpR medium is slowly removed with a capillary tube connected to a peristaltic pump, leaving behind 75 to I00/.d. Next, the protoplasts are gently resuspended by moving the tube between thumb and fingers, and transferred to an Eppendorf tube by using a wide-mouthed 200- or 1000-~1 tip pipettor. After addition of 0.25 vol (about 30 /~1) of a 4 x extraction buffer, the protoplasts are immediately chilled on ice. The protoplasts are broken by sonication for 5 to 6 sec. The sonicator probe should be set just below the surface of the liquid, and the amplitude set at 10 to 15/zm from peak to peak. To remove the cell debris, centrifuge the Eppendorf tubes at 4° for 1 min at ->9000 g in an Eppendorf centrifuge and withdraw the supernatant (containing the NPTII protein) to a precooled Eppendorf tube; store on ice. To compare the NPTII activity in different samples, equal amounts of cellular proteins are used in the NPTII assay. The protein content may be measured by the Bio-Rad (Richmond, CA) protein assay. Five microliters of each sample is mixed with 800/xl distilled water and 200/xl Bio-Rad dye, and transferred to a spectrophotometer cuvette. Before measuring the absorbance at 595 nm the reaction is allowed to proceed for 5 to I0 min. Absorption of the sample should be compared to a blank containing 5/xl of extraction buffer. If desired, a standard curve can be obtained using different amounts of bovine serum albumin (1 to 20/xg). For each NPTII determination, transfer a similar amount of protein to a precooled Eppendorf tube (we routinely load 50/zg of protein), and mix with 0.1 vol of 10 x loading buffer before loading on the nondenaturing protein gel. Preparation of Nondenaturing Gel. To prepare the protein gels, glass plates (15 x 17 cm) are thoroughly cleaned with detergent, then rinsed successively with tap water and ethanol, and dried (avoid contact between plates and skin). The plates are assembled with appropriate spacers (thickness, 1 mm) and clamps and, if necessary, sealed at the outside with a 1% (w/v) agarose solution. Next, the separation gel is poured between the glass plates up to 5 cm from their top. After carefully covering the gel solution with 0.5 cm of distilled water, the gel is allowed to polymerize (approximately 30 min at room temperature). Then the water is removed and the stacking solution is poured between the glass plates. A comb (thickness, 1 mm) with wells that can contain 100/xl of sample is inserted (avoid trapping air bubbles under the comb) and the gel is allowed to polymerize. After gently removing the comb and the bottom spacer, the gel is placed in an electrophoresis tank filled with running buffer and cooled for I hr before loading. The samples are loaded into the wells and run at 4-10 ° at 15 V/cm until the bromphenol blue marker has migrated 10 cm
438
REPORTER GENES
[37]
into the separation gel (approximately 5 hr). Then the two glass plates are laid horizontally and gently separated. Using a scalpel blade, the upper 10 cm of the separation gel is isolated and given three 15-min washes (with shaking) in 300 ml freshly made reaction buffer (chilled at 4°). Phosphotransferase Assay. Meanwhile, 500 mg of agarose (Sigma type II) is added to 50 ml reaction buffer, rapidly heated in a microwave oven to dissolve the agarose, and cooled to 45 ° in a water bath. Subsequently, in a disposable beaker or tube, add 10/zl [T-32p]ATP (100 ~Ci, 3000 Ci/ mmol) and 100/xl of a freshly prepared aqueous kanamycin sulfate stock (50 mg/ml) to 30 ml of the agarose and mix thoroughly. Lay the rinsed separation gel into a matching tray or box, and let it equilibrate to room temperature. Next, pour the agarose solution evenly over the separation gel. Wait for the gel to solidify and cover first with one sheet of Whatman (Clifton, N J) P8 ! paper and then with two sheets of Whatman 3MM paper (all cut to size and prewet in reaction buffer), a 5-cm stack of paper towels (cut to size), and a 1-kg weight evenly distributed over the top of the towels. After 3 to 12 hr of blotting, discard the paper towels and remove the Whatman P81 paper. Wash this four times with 200 ml of washing solution for 15 min each, at 80°. The Whatman P81 paper is then covered with thin plastic wrap, and exposed to X-ray film (XAR; Kodak, Rochester, NY) using an intensifying screen at - 70°. Exposure time varies from 10 min to 72 hr, depending on the strength of the NPTII signal. As controls for the NPTII assay, we used protein extracts from an untransformed tobacco plant (blank) and a tobacco plant transformed with the P2'-nptII construct (positive control). NPTII activity is detected by production and binding of phosphorylated kanamycin to the Whatman P81 paper. One often sees two slower migrating bands above this that are also detected in untransformed material and probably correspond to phosphorylated proteins. To quantitate the NPTII activity, the autoradiogram is matched with the Whatman P81 paper, the NPTII spots are located, cut out with a scalpel blade, air dried, submerged in scintillation liquid, and counted in a Beckman (Palo Alto, CA) scintillation counter.
Conclusions Of the six promoters tested in protoplasts, and the four in leaf bases (Table IV), the two strongest and least influenced by the source of protoplast or explant are derived from the mannopine synthase 2' gene and the cauliflower mosaic virus (CaMV) 35S transcript. It is useful to note that the ratio of activities of the P35S and Pnos constructs measured in transient expression assays 23 (approximately 3.6) is similar to the ratio of transfor-
[37]
SELECTABLE
MARKERS
439
FOR RICE TRANSFORMATION
T A B L E IV RELATIVE N P T I I ACTIVITY (%) PRODUCED BY DIFFERENT CHIMERIC GENES IN PROTOPLASTS AND LEAF BASESa Protoplasts from P r o m o t e r fused to nptlI gene (reference) N o n e (23, 26) P2' (23, 26)
P35S (23, 26) Pnos (23) PI' (23) Pz4 (23) P4.7 (23) P e x t e n s i n (26) Pcab (26)
E x p l a n t s from
Suspension cultures
Leaves
L e a f bases (L/D) b
Leaves (L/D)
ND 100 40 11 10 0.4 0.2 ---
ND 100 -8 -0.4 0.2 ---
ND/ND 93/100 95/98 ------/6 ND/ND
ND/ND 20/100 26/96 -----83/ND
" In each column, all e x p e r i m e n t s have been normalized to P 2' -dri ve n N P T I I activity. ( R e p r o d u c e d from D e k e y s e r et al. 26 with permission by the A m e r i c a n Society of Plant Physiologists.) b L e a f bases or leaves have been p r e g r o w n and maintained in light (L) or da rk (D) for 4 days after electroporation. ND, Not detected.
mation frequencies obtained with each promoter under selective conditions (2.6 and 3.0). 6 This indicates transient assays measure parameters that are useful for predicting transformation efficiencies. We have measured the growth rates of calli containing each of four different selectable markers (Pnos : nptII, P2' : nptII, P35S : bar, and P l ' : d h f r ) on different concentrations of the corresponding selective agents. These calli contained similar numbers of inserts, 23 and as seen in Table III were isolated at comparable frequencies, although some were selected on 200/xg kanamycin/ml, and some with 10/xg phosphinothricin/ ml. Similarly, there were comparable levels of NPTII activity in the calli analyzed, irrespective of how they were initially selected. 23 The first relevant observation made on the regenerate material was that there was no significant difference in the growth rates of transformed and untransformed calli on 0, 100, or 500/zg kanamycin/ml. On the other hand, the same transformed lines grew 8-10 times faster than untransformed calli on both 100 and 500 /xg G418/ml, for which rice has no endogenous resistance. Thus, to minimize the frequency of escapes from selection, one should use G418 instead of kanamycin whenever possible. Calli containing the P35S : bar construct also grew well under selection, in fact, 10-15 times faster than untransformed calli on 10 or 50 /xg of
440
REPORTER GENES
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phosphinothricin/ml. Calli containing PI' : dhfr did not grow rapidly on either 2 or I0/~g methotrexate/ml. All untransformed material died within 1 week. Either of these selection systems could, therefore, be used in place of the aminoglycoside detoxification system. However, it must be noted that the majority of fertile, transgenic plants obtained to date have been isolated using P35S : h p t 3'5'6'29
Variables in Gene Expression in Transformed Material The expression of transferred genes often varies from transformant to transformant. Sometimes the new genes are poorly expressed in some lines, or even silent. 29'3° In other cases, apparently homogeneous calli do not express fl-glucuronidase activity uniformly,3° or NPTII-deficient plants regenerate from NPTII-positive material, z A priori, these phenomena might indicate that the originally selected colonies were in fact polyclonal; however, other factors beyond the control of the investigator might produce similar results. For example, gene expression can be reduced by changes in DNA methylation or organization occurring in response to tissue culture conditions. 3L32 In other cases, activation or inactivation of nearby chromosomal promoters could silence gene transcription or generate antisense transcripts to reduce the expression of the foreign gene. On the other hand, foreign gene expression in most calli stabilizes within 3 to 5 months of culture, 8 and can remain constant in the absence of selection.Z,3,5,6, 30
Although the presence of the selectable marker can sometimes be verified by assaying for the corresponding enzyme activity, the presence of the cotransferred genes that have no overt phenotype can be ascertained only by performing Southern hybridization. 33 To answer questions about the organization of the new genes, one should always use restriction enzymes with targets that are generally not methylated in eukaryotes such as BgllI, EcoRI, or HindlII. Preferably, at least one (or a mixture of two) of the enzymes should be chosen because it cuts twice within the gene under investivation and generates a fragment that must be intact for the gene to function. In appropriate reconstruction experiments, one can compare the intensity of hybridization of probes to genomic DNA and to 29 R. Terada and K. Shimamoto, Mol. Gen. Genet. 220, 389 (1990). 3o W. Zhang and R. Wu, Theor. Appl. Genet. 76, 835 (1988). 31 p. T. H. Brown, J. Kyozuka, Y. Sukekiyo, Y. Kimura, K. Shimamoto, and H. L6rz, Mol. Gen. Genet. 223, 324 (1990). 32 E. MOiler, P. T. H. Brown, S. Hartke, and H. LOrz, Theor. Appl. Genet. 80, 673 (1990). 33 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980.
[38]
THAUMATIN II
441
similarly cut plasmid to estimate the copy number of the foreign gene. A single-copy equivalent of a 10-kb sequence in rice, assuming a diploid genome size of 4 x 108 bp, is approximately 2.5 × 10 -5/xg//zg genomic DNA. Similar analyses with a second restriction enzyme that cuts once, or not at all, in the transferred D N A can clarify the number of independent integration sites of the gene in the host sequence. We have analyzed seven transformed rice callus lines using BgllI, EcoRI, and a mixture of EcoRV and BstEII. Forty percent had single copies of the selected markers. Neither these transformants, nor others listed in Table IV, showed a consistent correlation between copy number and either gene expression or levels of resistance. Because deletions and mutations may be c o m m o n during integration of foreign sequences, it is possible many of these genes are not expressed. Acknowledgments This work was supported by grants from the Rockefeller Foundation (RF 86058 #59) and the Services of the Prime Minister (U.I.A.P. #120CO187). R.D. was a Research Assistant of the National Fund for Scientific Research (Belgium).
[38] T h a u m a t i n
II: A S w e e t M a r k e r
G e n e f o r U s e in P l a n t s
By MICHAEL WITTY Introduction Thaumatin II is a natural product of the West African plant Thaumatococcus daniellii. The fruit of this plant produces a family of five or more extremely sweet proteins ~called thaumatins that are traditionally used by West Africans to sweeten food and beverages. 2 In 1839 DanieU, the first European to write ofthaumatin, described it as having " . . . anindescribable yet intense degree of dulcidity . . . . ,,2 Thaumatin has long been a substance well known for the intensity of its taste and the small amount needed to produce an obvious sensation. Thaumatin solutions taste sweet at concentrations as low as 10 -8 M . 3 Thaumatins also lower the taste
l H. van der Wel and K. Loeve, Eur. J. Biochem. 31, 221 (1972). 2 W. F. Daniell, Pharm. J. 14, 158 (1855). 3 j. D. Higginbotham, in " D e v e l o p m e n t s in Sweeteners I , " p. 87. Applied Science Publ., London, 1979.
METHODS IN ENZYMOLOGY, VOL. 216
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