into germinating pollen by electroporation (9), including Southern blo t analysis to .... field strengths lead to irreversible breakdown of the cell membranes . 3.
CHAPTER 6
Pollen Electrotransformation in Tobacco James A. Saunders and Benjamin F. Matthew s 1 . Introductio n Numerous techniques have been developed to transfer genes int o plants to create genetically engineered crops that can tolerate environmental stresses, and to improve productivity and quality . The search fo r easier, more efficient techniques to transfer genes continues because th e efficiencies of current techniques are low and recovering fertile trans genic plants is difficult and time consuming with some plant species . Stable transformation of plant cells has been achieved using a numbe r of different mechanisms for DNA uptake . Transforming pollen wit h genetically engineered genes and using this pollen to fertilize flowers t o produce genetically engineered seed is one promising research area fo r obtaining transgenic plants faster and easier than some previous proce dures . This transformation approach is beginning to receive more atten tion because it circumvents the need for tissue culture, which require s extensive facilities for maintenance and manipulation of sterile explants . It also avoids the time-consuming task of regenerating whole plants from transformed protoplasts or plant tissues, which can take several months , require intensive labor, and expensive facilities . Often, because of th e many months of tissue culture required to regenerate plants, many of th e regenerated, transgenic plants are infertile and produce no seed, thus fur ther delaying the program . Pollen transformation also bypasses the use of Agrobacterium tumefaciens, which is commonly used to produce transgenic plants of tobacc o and several other plants . A . tumefaciens works well with a number o f From : Methods in Molecular Biology, Vol. 55: Plant Cell Electroporatio n and Electrofusion Protocols Edited by : J . A . Nickoloff Humana Press Inc ., Totowa, N J
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plants and is a routine procedure in many laboratories, however it has a limited host range and does not efficiently transform all plant types. Pollen transformation does not require A . tumefaciens, therefore is not limited to the A . tumefaciens host range, being limited only to the broade r range of plants reproducing via pollen. The underdeveloped technology of pollen transformation has the potential to produce a large variety o f transgenic plants in species previously difficult to genetically engineer . The concept of the use of pollen to effect genetic modification of subsequent progeny has been cited in the literature for some time and th e term pollen transformation was coined in the 1970s (1) . Several research ers have suggested that DNA, when added to pollen in either a solutio n or a paste, is capable of being taken up and expressed in progeny . For example, De Wet et al . (2) and Ohta (3) both using corn, indicated tha t pollen treated with exogenously added DNA could fertilize flowers an d produce seed that phenotypically expressed characteristics of the foreig n DNA. Pandey (4,5) described the sterilization of pollen of Nicotiana by X-ray irradiation and successful pollination of flowers with these treate d samples . This report provided evidence that pollen transformation ma y be functional using the denucleated pollen as a DNA vector . In addition , Hess (1) described a series of reports using petunia and corn that indicate that some DNA uptake may occur in pollen exposed to exogenousl y added DNA . Unfortunately, none of these reports proposed any mechanism for introducing the DNA into the pollen, nor did they obtain conclusive molecular evidence that gene transfer actually occurred. These deficiencies were pointed out by Sanford et al . (6) and others who were unable to repeat the results of these pioneer studies and th e process of pollen transformation had been left in a state of skepticism . Additional complications were added by Matousek and Tupy (7) an d Roeckel et al . (8), who described very active DNA nucleases that wer e present on the pollen wall and were released in an active state after pollen germination . These studies suggest that DNA would be degrade d within a few minutes if present in a mixture with germinating pollen . As a result of these negative reports, research on pollen transformatio n declined, waiting for a mechanism to be found to incorporate exogenously supplied DNA into the pollen grain rapidly enough so that it i s not degraded by nucleases . Results from our laboratory indicate that electroporation is an effective mechanism for transferring DNA rapidly int o germinating pollen .
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Our laboratory reported methods for incorporating radiolabeled DN A into germinating pollen by electroporation (9), including Southern blo t analysis to confirm DNA uptake (10) . Also, we reported the production of transgenic tobacco plants from pollen containing either the gene encodin g (3-glucuronidase (GUS) or chloramphenicol acetyltransferase (CAT ) (11) . The transgenic nature of these plants was confirmed by the presenc e of DNA encoding marker genes as detected by Southern hybridizatio n and by PCR amplification and hybridization . Expression of GUS activity measured by fluorometric assay and the visualization of GUS activity b y histological staining provided further evidence that these were transgeni c plants . In addition, it was demonstrated that in tobacco endogenou s nuclease activity can be reduced to acceptable levels by washing the pollen with fresh media after germination and immediately prior to the electroporation treatment (12) . Here we report the details of the protocols fo r pollen electrotransformation . 2. Material s 1. Pollen is collected from tobacco (Nicotiana gossei L. Domin) plants grown i n the greenhouse under natural light supplemented with fluorescent light t o achieve a 16-h photoperiod or from field grown plants . Tobacco pollen i s collected in the morning and used the same day; however, we have stored the pollen at -70°C for up to 6 wk without serious decreases in pollen viability . 2. Germination medium (GM ; 13): 10% (w/v) sucrose, 1 .27 mMCa(NO3)2, 0.1 6 mM H3B O3 , and 1 mM KN O3 , pH adjusted to 5 .2 with additional borate . 3. Electroporation equipment : square wave generator (e .g., BTX model 200, BTX Inc ., San Diego, CA) . 4. The GUS reporter gene construct pBI221 is a 5 .7-kbp plasmid (ClonTech, Palo Alto, CA) . Linearize the plasmid with EcoRI prior to electroporation to facilitate incorporation . 5. Gibberellic acid (GA3) : Dissolve in ethanol as a stock solution of 100 .g/mL and store at -10°C until use . p 6. Plant seeds in 5-in . pots containing Metro-Mix 500 (Grace Sierra Horticultural Products Co ., Milpitas, CA) and soil mixed 1 :1 ; water daily . 7. Trypan blue solution : 1 mg/mL in 0 .6M mannitol . 8. Fluorescein diacetate (FDA) : Prepare a stock solution of 1 mg/mL i n acetone. Dilute the stock solution (1 :5) with 0 .6M mannitol for use . The diluted working solution is only stable for 1 h before the dye precipitate s out of solution . 9. A fluorescent microscope equipped with excitation wavelength filter o f 485 nm and an emission filter of 520 nm .
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1 . Germinate pollen at a concentration of 4 mg/300 µL in GM in a 2-m m disposable electroporation chamber (e .g., Model 620, BTX Inc .) for 1 h at 30°C in a rotary shaker (50 rpm) . 2. Change GM immediately prior to the electroporation to remove an y endogenous nucleases that may be released from the pollen wall durin g germination (7,12,14) . The GM can be removed by gentle aspiration afte r either gravity sedimentation of the pollen or after the pollen is pelleted b y centrifugation at 50g in a horizontal rotor for 1 min . 3 . Add the linearized DNA of interest at a final concentration of 10—2 0 p.g/mL, immediately before the electroporation pulse . 4 . Typically pollen treatments and controls include : a. No electroporation in the presence of DNA ; b. Electroporation with DNA ; and c. Electroporation without DNA . 5. Electroporate the pollen by either a single or dual pulse from a square or exponential pulse generator (see Note 1) . It is best to examine a range of field strengths from 0—10 kV/cm in 0 .5-kV increments . For tobacco pollen, a square wave pulse duration of 80 µs and a cuvet with a 1-mm electrode gap can be used . The optimal conditions for transformation of th e germinating tobacco pollen with DNA employs a single 8 .75 kV/cm, 80 µs square wave pulse . Alternatively, an exponential pulse generator (BT X Model 600) can be used to achieve efficient pollen electrotransformatio n in germinating tobacco pollen . We tested exponential pulses from 0—1 0 kV/cm in 0 .5-kV increments using a cuvet with a 2-mm electrode gap at a resistance of 360 S2 . With this system, successful incorporation of DN A was accomplished between 4 and 6 kV/cm while maintaining high pollen viability (see Note 2) . The range of successful pulse field strengths for the exponential pulse generator is slightly narrower than that of the squar e wave generator (see Note 3) . 6. Following electroporation, gravity sediment the pollen within the electroporation cuvet for 10 min . Very carefully remove most of the electroporation medium without disturbing the pollen using a pipet . 7. Emasculate recipient flowers by removing the anthers and bagging th e inflorescence 4 d before the electroporation treatment to prevent pollination . Emasculation of flowers prior to electroporation greatly enhances th e production of viable seed produced through fertilization with electroporated pollen (see Note 4) .
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8. Pollinate the stigmas of emasculated flowers using the concentrated polle n slurry . Pollinate flowers by pipeting 10—20 µL of the pollen onto the top o f the stigma . Rebag the flower for seed production . In some species, such a s corn or alfalfa, it may be easier to scoop the pollen slurry out of the electroporation cuvet with a spatula . In any case, use extreme care to preven t damaging the fragile pollen tubes . 9. Collect seeds from pollen-treated flowers and determine seed number , viability, and the number of plants showing positive expression of th e introduced trait . Putative transformants that yield a positive expressio n assay can be verified by Southern hybridization and/or PCR amplificatio n of the GUS coding region of the pBI221 plasmid (11) . 3 .2.
Seed Processing and Planting
1. Surface sterilize seeds obtained from electroporated pollen-treated flower s with 10% (v/v) commercial bleach . Incubate seeds in 200 tL of GA 3 at a concentration of 10 µg/mL for 10 min to release them from dormancy . 2. Plant the seeds in Metro-Mix 500 and soil on a misting bench in the gree n house and grow until the plants are large enough to be transplanted individually into 5 in . clay pots . 3.3. Optimizing Electroporation Condition s
with Cytochemical Stains : Viability and Uptake Assay It is desirable to examine a range of electroporation conditions whe n pollen from different species or varieties are used . This can be quit e tedious when doing expression assays, particularly with plants produce d from seed of treated pollen that had been electroporated weeks or month s earlier . The combination of two cytochemical stains, trypan blue an d fluorescein diacetate (FDA), can be used to rapidly determine the optimal electroporation conditions for DNA uptake while maintaining pollen viability (15) . 1. Electroporate 200 µL aliquots of germinated pollen with 20 µL of trypa n blue solution . 2. Immediately observe the cells under a bright field microscope and scor e for the uptake of the blue dye . Blue color indicates that the cell membrane of the protoplasts has been permeated by the electroporation pulse, how ever, it does not distinguish between cells that are alive and those that hav e been killed by the electroporation treatment . 3. An aliquot of cells from the same population should be electroporated with out trypan blue and stained for viability with an equal volume of 0 .1 mg/mL
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Saunders and Matthew s FDA in 0.6Mmannitol after a 1-h incubation at room temperature . Brigh t yellow-green fluorescence of cells indicates a positive viability assay wit h a fluorescent microscope as described previously (16,17). Examine at leas t three replicates of 100 cells by light microscopy and score for both viability using FDA and uptake with trypan blue . The working range for electroporation conditions is that where trypan blue uptake has occurred, bu t without excessive loss of cell viability . Generally this is the point on th e graph where the two lines intersect . 4. Note s
1. In general, two different DC high voltage pulse wave forms can be utilize d to transform pollen, the square wave pulse and the exponential wave pulse . Using the square wave pulse, both the amplitude and the duration of the pulse can be accurately controlled . With the exponentially decaying pulse , the amplitude of the wave form can be accurately controlled, however, th e duration of the pulse can only be modified in a general manner . As i n the case of plant protoplasts (18,19), animal cells (20-22), and yeast (23) , the success and efficiency of introducing DNA or RNA into the germinating pollen by electroporation depends on several important variable s (9,10,24), including the pulse field strength, the pulse duration, the resealing time of the pores introduced into the cell membrane, and the concentration of pollen and DNA in the electroporation medium (25) . 2. The field strength of the pulse is controlled by two components : the applied current and the electrode gap . To have an effective electroporation pulse , minimal threshold levels for both the pulse duration and the pulse fiel d strength must be exceeded . Our data suggest that the field strength o f the pulse interacts with pulse duration such that, over a limited range, on e variable may be increased as the other is decreased and a reversible por e may still be induced . Liang et al . (26) have suggested that pore induction , size, and frequency are controlled by pulse height, whereas the length o f time the pores remain open is controlled by pulse duration . Benz and Zimmerman (27), have indicated that different cell types require differen t field strengths to induce pores because of differences in membrane composi tion and osmotic properties . Pulses that do not meet minimum field strengt h thresholds of durations may not induce any pore formation, and excessive field strengths lead to irreversible breakdown of the cell membranes . 3. We recommend testing a broad range of pulse field strengths whe n attempting the electroporation of a new population of cells . This is t o determine exactly the pulse field strength sufficient to cause DNA uptak e and to determine if the pulse field strength is causing unacceptable damag e to the cell viability . Although we do not recommend the procedure, there is
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an understandable tendency on the part of many investigators to electroporate their cell lines at published pulse field strengths without checkin g uptake and viability . Without an accurate knowledge of the effects of th e electroporation pulse on the viability of the cells being used, a square wav e pulse of
