CHAPTER 6

Pollen Electrotransformation in Tobacco

James A. Saunders and Benjamin F. Matthews

1. IntroductionNumerous techniques have been developed to transfer genes int o

plants to create genetically engineered crops that can tolerate environ-mental stresses, and to improve productivity and quality. The search fo reasier, more efficient techniques to transfer genes continues because th eefficiencies 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 rof different mechanisms for DNA uptake . Transforming pollen wit hgenetically engineered genes and using this pollen to fertilize flowers t oproduce genetically engineered seed is one promising research area fo robtaining 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 sextensive facilities for maintenance and manipulation of sterile explants .It also avoids the time-consuming task of regenerating whole plants fromtransformed protoplasts or plant tissues, which can take several months ,require intensive labor, and expensive facilities . Often, because of themany months of tissue culture required to regenerate plants, many of th eregenerated, transgenic plants are infertile and produce no seed, thus fur -ther delaying the program .

Pollen transformation also bypasses the use of Agrobacterium tume-faciens, which is commonly used to produce transgenic plants of tobacc oand several other plants . A . tumefaciens works well with a number o f

From : Methods in Molecular Biology, Vol. 55: Plant Cell Electroporatio nand 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 alimited host range and does not efficiently transform all plant types. Pol-len transformation does not require A . tumefaciens, therefore is not lim-ited to the A . tumefaciens host range, being limited only to the broade rrange of plants reproducing via pollen. The underdeveloped technologyof pollen transformation has the potential to produce a large variety o ftransgenic plants in species previously difficult to genetically engineer .

The concept of the use of pollen to effect genetic modification of sub-sequent progeny has been cited in the literature for some time and th eterm pollen transformation was coined in the 1970s (1) . Several research -ers have suggested that DNA, when added to pollen in either a solutio nor a paste, is capable of being taken up and expressed in progeny . Forexample, De Wet et al . (2) and Ohta (3) both using corn, indicated thatpollen treated with exogenously added DNA could fertilize flowers an dproduce seed that phenotypically expressed characteristics of the foreig nDNA. Pandey (4,5) described the sterilization of pollen of Nicotiana byX-ray irradiation and successful pollination of flowers with these treatedsamples . This report provided evidence that pollen transformation ma ybe functional using the denucleated pollen as a DNA vector . In addition ,Hess (1) described a series of reports using petunia and corn that indicatethat some DNA uptake may occur in pollen exposed to exogenousl yadded DNA. Unfortunately, none of these reports proposed any mecha-nism for introducing the DNA into the pollen, nor did they obtain con-clusive molecular evidence that gene transfer actually occurred.

These deficiencies were pointed out by Sanford et al . (6) and otherswho were unable to repeat the results of these pioneer studies and th eprocess of pollen transformation had been left in a state of skepticism .Additional complications were added by Matousek and Tupy (7) an dRoeckel et al . (8), who described very active DNA nucleases that wer epresent on the pollen wall and were released in an active state after pol-len germination . These studies suggest that DNA would be degradedwithin a few minutes if present in a mixture with germinating pollen . Asa result of these negative reports, research on pollen transformatio ndeclined, waiting for a mechanism to be found to incorporate exog-enously supplied DNA into the pollen grain rapidly enough so that it i snot degraded by nucleases . Results from our laboratory indicate that elec-troporation is an effective mechanism for transferring DNA rapidly intogerminating pollen .

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Our laboratory reported methods for incorporating radiolabeled DN Ainto germinating pollen by electroporation (9), including Southern blo tanalysis to confirm DNA uptake (10) . Also, we reported the productionof 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 eof DNA encoding marker genes as detected by Southern hybridizatio nand by PCR amplification and hybridization. Expression of GUS activitymeasured by fluorometric assay and the visualization of GUS activity b yhistological staining provided further evidence that these were transgenicplants . In addition, it was demonstrated that in tobacco endogenou snuclease activity can be reduced to acceptable levels by washing the pol-len with fresh media after germination and immediately prior to the elec-troporation treatment (12) . Here we report the details of the protocols fo rpollen electrotransformation .

2. Materials

1. Pollen is collected from tobacco (Nicotiana gossei L. Domin) plants grown i nthe greenhouse under natural light supplemented with fluorescent light t oachieve a 16-h photoperiod or from field grown plants . Tobacco pollen i scollected in the morning and used the same day; however, we have stored thepollen 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(NO 3) 2 , 0 .1 6mM H 3BO3 , and 1 mM KNO 3 , 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 electroporationto facilitate incorporation .

5. Gibberellic acid (GA3 ) : Dissolve in ethanol as a stock solution of 100p.g/mL and store at -10°C until use .

6. Plant seeds in 5-in . pots containing Metro-Mix 500 (Grace Sierra Horticul-tural 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 . Thediluted working solution is only stable for 1 h before the dye precipitate sout of solution .

9. A fluorescent microscope equipped with excitation wavelength filter o f485 nm and an emission filter of 520 nm .

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3. Methods

3.1. Pollen Electroporation1 . 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 at30°C in a rotary shaker (50 rpm) .

2 . Change GM immediately prior to the electroporation to remove an yendogenous nucleases that may be released from the pollen wall durin ggermination (7,12,14) . The GM can be removed by gentle aspiration afte reither gravity sedimentation of the pollen or after the pollen is pelleted b ycentrifugation at 50g in a horizontal rotor for 1 min .

3 . Add the linearized DNA of interest at a final concentration of 10—2 0p.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; andc. Electroporation without DNA.

5. Electroporate the pollen by either a single or dual pulse from a square orexponential pulse generator (see Note 1) . It is best to examine a range offield strengths from 0—10 kV/cm in 0 .5-kV increments . For tobacco pol-len, a square wave pulse duration of 80 µs and a cuvet with a 1-mm elec-trode gap can be used . The optimal conditions for transformation of th egerminating tobacco pollen with DNA employs a single 8 .75 kV/cm, 80 µssquare wave pulse. Alternatively, an exponential pulse generator (BT XModel 600) can be used to achieve efficient pollen electrotransformationin germinating tobacco pollen . We tested exponential pulses from 0—1 0kV/cm in 0 .5-kV increments using a cuvet with a 2-mm electrode gap at aresistance of 360 S2 . With this system, successful incorporation of DNAwas accomplished between 4 and 6 kV/cm while maintaining high pollenviability (see Note 2) . The range of successful pulse field strengths for theexponential pulse generator is slightly narrower than that of the squar ewave generator (see Note 3) .

6 . Following electroporation, gravity sediment the pollen within the elec-troporation cuvet for 10 min . Very carefully remove most of the electropo-ration medium without disturbing the pollen using a pipet .

7 . Emasculate recipient flowers by removing the anthers and bagging th einflorescence 4 d before the electroporation treatment to prevent pollina-tion. Emasculation of flowers prior to electroporation greatly enhances th eproduction of viable seed produced through fertilization with electro-porated pollen (see Note 4) .

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8. Pollinate the stigmas of emasculated flowers using the concentrated polle nslurry . Pollinate flowers by pipeting 10—20 µL of the pollen onto the top o fthe stigma. Rebag the flower for seed production . In some species, such a scorn or alfalfa, it may be easier to scoop the pollen slurry out of the elec-troporation cuvet with a spatula . In any case, use extreme care to preven tdamaging 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 eintroduced trait . Putative transformants that yield a positive expressionassay can be verified by Southern hybridization and/or PCR amplificatio nof the GUS coding region of the pBI221 plasmid (11) .

3.2. Seed Processing and Planting1. Surface sterilize seeds obtained from electroporated pollen-treated flower s

with 10% (v/v) commercial bleach . Incubate seeds in 200 tL of GA3 at aconcentration 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 nhouse and grow until the plants are large enough to be transplanted indi-vidually into 5 in . clay pots .

3.3. Optimizing Electroporation Condition swith Cytochemical Stains: Viability and Uptake AssayIt is desirable to examine a range of electroporation conditions whe n

pollen from different species or varieties are used . This can be quit etedious when doing expression assays, particularly with plants produce dfrom seed of treated pollen that had been electroporated weeks or month searlier. The combination of two cytochemical stains, trypan blue an dfluorescein diacetate (FDA), can be used to rapidly determine the opti-mal electroporation conditions for DNA uptake while maintaining pol-len viability (15) .

1. Electroporate 200 µL aliquots of germinated pollen with 20 µL of trypa nblue solution .

2. Immediately observe the cells under a bright field microscope and scor efor the uptake of the blue dye . Blue color indicates that the cell membraneof the protoplasts has been permeated by the electroporation pulse, how -ever, it does not distinguish between cells that are alive and those that havebeen 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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FDA in 0.6Mmannitol after a 1-h incubation at room temperature . Brigh tyellow-green fluorescence of cells indicates a positive viability assay wit ha fluorescent microscope as described previously (16,17) . Examine at leas tthree replicates of 100 cells by light microscopy and score for both viabil-ity using FDA and uptake with trypan blue. The working range for elec-troporation conditions is that where trypan blue uptake has occurred, bu twithout excessive loss of cell viability . Generally this is the point on th egraph where the two lines intersect .

4. Notes

1. In general, two different DC high voltage pulse wave forms can be utilize dto transform pollen, the square wave pulse and the exponential wave pulse .Using the square wave pulse, both the amplitude and the duration of thepulse can be accurately controlled . With the exponentially decaying pulse ,the amplitude of the wave form can be accurately controlled, however, theduration of the pulse can only be modified in a general manner . As i nthe case of plant protoplasts (18,19), animal cells (20-22), and yeast (23) ,the success and efficiency of introducing DNA or RNA into the germinat-ing pollen by electroporation depends on several important variable s(9,10,24), including the pulse field strength, the pulse duration, the reseal-ing time of the pores introduced into the cell membrane, and the concen-tration of pollen and DNA in the electroporation medium (25) .

2. The field strength of the pulse is controlled by two components : the appliedcurrent and the electrode gap . To have an effective electroporation pulse ,minimal threshold levels for both the pulse duration and the pulse fieldstrength must be exceeded . Our data suggest that the field strength ofthe pulse interacts with pulse duration such that, over a limited range, on evariable may be increased as the other is decreased and a reversible por emay still be induced . Liang et al . (26) have suggested that pore induction ,size, and frequency are controlled by pulse height, whereas the length o ftime the pores remain open is controlled by pulse duration . Benz andZimmerman (27), have indicated that different cell types require differen tfield strengths to induce pores because of differences in membrane composi -tion and osmotic properties . Pulses that do not meet minimum field strengt hthresholds of durations may not induce any pore formation, and excessivefield strengths lead to irreversible breakdown of the cell membranes .

3. We recommend testing a broad range of pulse field strengths whe nattempting the electroporation of a new population of cells . This is t odetermine exactly the pulse field strength sufficient to cause DNA uptak eand to determine if the pulse field strength is causing unacceptable damag eto 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 electro-porate their cell lines at published pulse field strengths without checkin guptake and viability . Without an accurate knowledge of the effects of th eelectroporation pulse on the viability of the cells being used, a square wav epulse of <100 is may yield more "forgiving" positive results over a broaderrange of pulse field strengths .

4 . The emasculation procedure was necessary to prevent pollination of th eflower by nontreated pollen . Typically, emasculation was performed b yplucking the anther sacs from the flower before flower anthesis, but defi-nitely before the anthers opened . There appeared to be a stimulatoryresponse to seed set with emasculation several days before pollination . Forexample, emasculation of the recipient flowers in Nicotiana gossei prior topollination with electroporated pollen resulted in a 10-fold increase in th enumber of seeds formed in the seed capsule (9) .

References1. Hess, D . (1987) Pollen-based techniques in genetic manipulation . Inter. Rev . Cytol.

107, 367-395 .2. De Wet, J . M . J ., Bergquist, R . R ., Harlan, J . F ., Brink, D . E ., Cohen, C . E ., Newell ,

C . A., and De Wet, A .-E . (1985) Exogenous gene transfer in maize (Zea mays)using DNA-treated pollen, in Experimental Manipulation of Ovule Tissue s(Chapman, G . P ., Mantell, S . H., and Daniels, R . W., eds .) Longman, London, pp .197-209 .

3. Ohta, Y . (1986) High-efficiency genetic transformation of maize by a mixture o fpollen and exogenous DNA . Proc . Natl . Acad. Sci . USA 83, 715-719 .

4. Pandey, K . K. (1978) Gametic gene transfer in Nicotiana by means of irradiatedpollen . Genetica 49, 53-69 .

5. Pandey, K. K. (1980) Further evidence for egg transformation in Nicotiana .Heredity 45, 15-29 .

6. Sanford, J . C ., Skubik, K . A., and Reisch, B . I . (1985) Attempted pollen-mediate dplant transformation employing genomic donor DNA . Theor. Appl. Genet. 69 ,571-574 .

7. Matousek, J . and Tupy, J . (1983) The release of nucleases from tobacco pollen .Plant Sci . Lett. 30, 83-89 .

8. Roeckel, P ., Heizmann, P ., Dubois, M ., and Dumas, C . (1988) Attempts to trans -form Zea mays via pollen grains, effect of pollen and stigma nuclease activities .Sex . Plant Reprod. 1, 156-163 .

9. Abdul-Baki, A. A., Saunders, J . A., Matthews, B . F., and Pittarelli, G . W. (1990 )DNA uptake by electroporation of germinating pollen grains . Plant Sci. 70, 181-190 .

10. Matthews, B . F ., Abdul-Baki, A. A., and Saunders, J . A. (1990) Expression of a for-eign gene in electroporated pollen grains of tobacco . Sex. Plant Reprod. 3, 147-151 .

11. Smith, C . R ., Saunders, J . A., Van Wert, S ., Cheng, J ., and Matthews, B . F. (1994 )Expression of GUS and CAT activities using electrotransformed pollen . Plant Sci.104, 49-58 .

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12. Van Wert, S . L . and Saunders J . A. (1992) Reduction of nuclease activity releasedfrom germinating pollen under conditions used for pollen electrotransformation .Plant Sci . 84, 11-16 .

13. Dickinson, D . B. (1968) Rapid starch synthesis associated with increased respira-tion in germinating lily pollen. Plant Phys. 43, 1-8 .

14. Matousek, J . and Tupy, J. (1984) Purification and properties of extracellula rnuclease from tobacco pollen . Bio. Plantarum 26, 62-73 .

15. Saunders, J . A., Lin, C . H., Cheng, J ., Tsengwa, N ., Lin, J . J ., Smith, C . R ., McIn-tosh, M., and Wert, S . V. (1994) Rapid optimization of electroporation condition sfor plant cells, protoplasts, and pollen . Mol . Biotechnol. (in press) .

16. Saunders, J . A., Roskos, L . A ., Mischke, B . S ., Aly, M., and Owens, L . D . (1986)Behavior and viability of tobacco protoplasts in response to electrofusion param-eters . Plant Physiol . 80, 117-121 .

17. Abdul-Baki, A . A. (1992) Determination of pollen viability in tomatoes . J. Am.Soc . Hort. Sci . 117(3), 473-476 .

18. Fromm, M., Taylor, L . P ., and Walbot, V . (1985) Expression of genes transferre dinto monocot and dicot plant cells by electroporation . Proc . Natl. Acad. Sci. USA82, 5824-5828 .

19. Fromm, M . E ., Taylor, L . P ., and Walbot, V . (1986) Stable transformation of maiz eafter gene transfer by electroporation . Nature 319, 791-793 .

20. Neumann, E ., Schaefer-Ridder, M., Wang, Y ., and Hofschneider, P . H. (1982)Inhibition of gene expression in plant cells by expression of antisense RNA . EMBO.J. 1, 841-845 .

21. Wong, T. K. and Neumann, E . (1982) Electric field mediated gene transfer .Biochem. Biophys . Res . Comm . 107, 584-587 .

22. Potter, H., Weir, L ., and Leder, P . (1984) Enhancer-dependent expression o fhuman K immunoglobulin genes introduced into mouse pre-B lymphocytes by elec -troporation . Proc . Natl. Acad. Sci. USA 81, 7161-7165 .

23. Weber, H ., Forester, W., and Jacob, H . E . (1981) Parasexual hybridization of yeast sby electric field stimulated fusion of protoplasts . Curr. Genet. 4, 165,166 .

24. Saunders, J . A., Matthews, B . F., and Van Wert, S . L. (1991) Pollen electro-transformation for gene transfer in plants, in Guide to Electroporation an dElectrofusion (Chang, D . C ., Chassy, B . M., Saunders, J . A., and Sowers, A . E . ,eds .), Academic, San Diego, CA, pp . 227-247 .

25. Saunders, J . A ., Matthews, B . F ., and Miller, P . D . (1989) Plant gene transfer usingelectrofusion and electroporation, in Electroporation and Electrofusion in CellBiology (Neumann, E ., Sowers, A ., and Jordan, C ., eds .) Plenum, New York, pp .343-354 .

26. Liang, H., Purucker, W . J ., Stenger, D . A., Kubiniec, R . T., and Hui, S . W. (1988)Uptake of fluorescence-labeled dextrans by 10T 1/2 fibroblasts following permeationby rectangular and exponential electric field pulses . BioTechniques 6, 550-558 .

27. Benz, R., Zimmermann, U., and Wecker, E . (1981) High electric fields effects on th ecell membranes of Halicystis parvula : a charge-pulse study . Planta 152, 314-318 .