A transformation system utilizing the normal fertilization cycle of maize is desirable since the protoplast system has limitations in not regenerating whole plants. Pollen seems to be a suitable vehicle with which to transfer foreign DNA into maize plants. Hess et al. (Z. Pflanzenphysiol. 74:52-63, 1974) consider pollen of Petunia and Nicotiana can take up foreign DNA during the germination phase and transfer it to the egg during fertilization. Maize pollen can be germinated in vitro and transferred to silks to effect fertilization (Raman et al., J. Hered. 71:311-314, 1980). Recently, Ohta (P.N.A.S. 83:715-719, 1986) obtained genetic evidence for the successful transformation of maize using pollen to transfer exogenous DNA to the embryo and endosperm.
The method used to attempt to transfer cloned DNA into maize plants was to germinate maize pollen from alcohol dehydrogenase null plants in the presence of a plasmid containing Adh genes, then transfer pollen to the silks of detasseled, Adh null plants. A modification of the Brewbaker and Kwack medium (Amer. J. Bot. 50:859-865, 1963) was used to germinate maize pollen and maintain pollen tubes intact for at least two hours. The medium consisted of 15% sucrose, 0.03% Ca(N03)2, 0.01% H3BO3; 0.02% MgS04; 0.01% KN03; 0.01% Tris; 0.7% Agarose, pH 6.5. Small pieces of dialysis tubing were placed on the surface of the solid medium some time before the pollen was applied. The dialysis tubing prevented germinated pollen grains from adhering to the agar medium and allowed easy transfer to the silks with a camel hair brush. DNA, as a supercoiled plasmid, contained either the Adh1-1F or Adh1-1S allele, each in combination with nopaline synthase as another marker gene. DNA was applied to the surface of the dialysis tubing at concentrations between 50-200 µg/ml. The effect of linearizing the plasmid DNA was also tested. Fresh pollen from Adh null plants (#256 = Adh1-0, Adh2-0; g25 = Adh1-0, Adh2 +) was sprinkled on the surface of the DNA solution over the dialysis tubing. After a minimum of 15 minutes germination the pollen was transferred to the silks of detasseled, Adh null plants in a "pollen free" glasshouse. The resulting seed was grown to maturity and tested enzymically for ADH activity in small scutellar slices.
The time of pollen germination on artificial medium
before transfer to silks affected the resulting seed set:
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Shorter germination times produced more seed but the pollen was in contact with DNA for a shorter time. The possibility of seed being produced by non-germinated pollen grains cannot be excluded. However, counts suggested that generally if pollen grains did not germinate within 30 minutes on artificial medium they failed to germinate.
Another method used to determine if fertilization was effected by germinated pollen was to fix silks (3 volumes ethanol: 1 volume acetic acid) with the attached pollen and stain with aniline blue. Aniline blue stains specifically for callose in the pollen tubes and allows the course of the tubes to be followed down the silks to the ovules. Using this method, pre-germinated pollen grains could be seen to be attached to the silks and penetrate down to the ovule.
The site at which DNA is "taken up" by germinating pollen is uncertain. Hess et al. (Z. Pflanzenphysiol. 74:371-376, 1974) believe the tips of Petunia pollen tubes are thin and permeable and permit the uptake of large molecules. Experiments were conducted to try to detect the site of uptake of DNA in germinated maize pollen. Staining with the DNA specific fluorochromes, DAPI and mithramycin (Coleman et al., J. Histochem. Cytochem. 29:959-968, 1981), failed to detect any differences in DNA levels in treated and untreated pollen. However, the stains were useful in following the path of migration of pollen nuclei in the tubes of fixed material during germination. Pollen grains were also germinated in the presence of 31S labelled DNA, fixed and autoradiographed. A very small percentage of pollen tubes showed the presence of silver grains which represent DNA molecules, down the length of the pollen tube. Whether the silver grains were located inside or outside the pollen tubes was not known.
Approximately 1500 seeds were tested for ADH activity after DNA treatment of Adh null pollen. Two seeds from Adh1-1S treated pollen showed possible Adh1-1S activity in one Adh null line, and another from Adh1-1F treated pollen showed Adh1-1F activity in a different Adh null line. After germination the three seedlings showed weak growth and did not survive. A number of seedlings produced from germinated pollen grains showed weak growth, and embryo rescue from fertilized ovules was carried out to overcome the problem. Several hundred small plants were regenerated in tissue culture and the roots tested for ADH activity. None of the plants showed ADH activity.
Although Adh genes may have been transferred into three maize plants via pollen, this method, which utilizes the "passive" uptake of DNA by germinating pollen, does not appear to be practical to obtain a reproducible, high frequency of transformation. Sanford et al. (Theor. Appl. Genet. 69:571-574, 1985) were also unable to obtain satisfactory frequencies of pollen mediated transformation in maize and tomato. Recent work by Matougek and Tupy (J. Plant Physiol. 119:169-178, 1985) showed that pollen from a number of plants, including maize, released nucleases when germinated on artificial media. The combined effects of nucleases degrading the Adh containing DNA and the low seed set produced by pre-germinated pollen are some of the likely causes of the low frequency of pollen mediated transformation in maize.
An approach aimed at mechanically delivering DNA efficiently into cells is now being attempted to obtain transformation in maize.
J.C. Waldron
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