As outlined in the first article, we have attempted to recover zygotes from male gametophytes that were first germinated on a suitable medium and then transferred to the female gametophyte, suitably juxtapositioned to maximize micropyle penetration by the tube and hopefully fertilization. We have been successful in employing the method outlined below:
(1) Explant mature, unpollinated ovaries (following a modified protocol of Gengenbach, Planta 134:91-93, 1977).
(2) Germinate pollen on medium (Can. J. Bot. 43:779, 1965) for 15-30 min or until pollen tube is desired length.
(3) Prepare ovary by cutting silk back to within 1 mm.
(4) Carefully cut a block of agar (from step 2) such that a gametophyte remains intact, shaving the edge nearest the tube tip so that contact with the cut end of the silk can be made.
(5) Juxtaposition male gametophyte to female. Incubate at 25o- 30o C for period of growth of caryopsis.
(6) Storage of caryopsis; normal planting of seed. Or, embryo explant to medium followed by transplant to soil.
Since most of our evidence is photographic, it will be recorded elsewhere. Our success rate is now 25 mature, fertile plants from 212 attempted juxtapositions. Undoubtedly experience and technical modification will improve this ratio. It appears that it is now feasible to employ any selection agent on pollen such that survival rates of 1x10-6 and lower are useful (and desirable). We attempted to extend this technology to field scale in 1979. Fresh pollen (or pollen stored at +50 C) from a variety of genetically marked cultivars was employed. Germination (approximately 30 min) was accomplished on the supplemented agar medium or on the surface of the same medium without agar.
Various preparative methods of the female tissue were attempted. 'Windows' were cut in the sides of bagged ears to reveal silks. Ultimately we opted for the simplest method--the normal preparation of a brush of silks. Contamination was a critical problem. Precautions were taken and we expect that contaminants can be genetically identified. A 2 m x 1 m x 1 m plastic covered, portable enclosure was built and deployed to several cultivars in the field during the pollinating season. Pollination attempts that were undertaken outside the enclosure were conducted during hours of least contamination (evening and night).
Delivery of the pollen plus tube was by Pasteur pipette or by spatula. Insofar as was possible, with the aid of a field microscope, only germinated pollen grains with tubes longer than one diameter were applied to the brush of silks. A variety of 'controls' were attempted. The most informative control, listed in Table 1, utilized the application of burst and non-germinated pollen grains from the same plates as were taken the germinated ones.
Table 1. 'Phenotypic success' from germinated pollen grains
|Treatment*||Ears attempted||Ears with kernels||Kernels (range)|
A = pollen germinated on agar, delivered by spatula;
female in enclosure
B = pollen germinated on liquid, delivered by pipette; female in enclosure
C = treatment B except female not in enclosure
D = treatment A except female not in enclosure
control = treatments A, B, C or D but only burst or non-germinated pollen grains used
Each female ear in the study was 'pollinated' on at least four different days, each time with a genotypically different pollen source. The data in Table 1 must be viewed with reservation. Plants from kernels from various ears are growing in the 1980 winter nursery and will be testcrossed for confirmation of genotypic identity.
From these two studies--in vitro and in situ--it appears that manipulation of the male gametophyte to permit the challenge of a selection pressure (physical, chemical or biological) followed by recovery of a surviving gametophyte is now feasible.
D. B. Walden and K. Raman
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