A method to separate germinated from ungerminated pollen grains

Envtlonmental and E~perimenlal Botany, Vol. 33, No. 3, pp. 415 421, 1993 0098 8472]93 $6.110+ 0.00 (l~ 1993 Pergamon Press Lid Printed in (3reat Blitain. A M E T H O D TO SEPARATE G E R M I N A T E D F R O M U N G E R M I N A T E D POLLEN GRAINS Y. HI ZHANG,* LYLE E. CRAKER*~ and DAVID L. MULCAHY* Departments of *Plant and Soil Sciences and ++Biology,University of Massachusetts, Amherst, MA 01003, U.S.A. (Received 15 September 1992; accepted in revisedform 20 November 1992) ZHANGY. H., CRAKERL. E. and MULCAHYD. L. A method to separategerminatedfrom ungerminated pollen grains. ENVIRONMENTALAND EXPERIMENTALBOTANY33, 415--421, 1993. A simple, nontoxic and effective method to separate germinated from ungerminated pollen grains has been developed using pollen from corn (Zea mays L. cv. Pioneer 3747). After separation, 99.1% of the grains in the sample of germinated pollen grains had tubes; 97.2°,{) of the grains in the sample of ungerminated pollen grains had no tubes. Recovery was 18.1% of the total grains for germinated and 1.5% for ungerminated pollen grains. The separated germinated pollen grains retained viability and continued tube growth when placed in culture medium. INTRODUCTION THE life-cycle of higher plants has two phases: a conspicuous sporophytic phase and a reduced gametophytic phase. While natural selection undoubtedly occurs in both phases, artificial selection for desired traits has historically been performed in the sporophytic phase. (9/ TERAVANESIAN (13A4) and MULCAHY (7) have proposed, however, that selection at the gametophytic phase is possible and could be applied in plant breeding. Studies with isoenzymes and nucleic acids indicate an overlap of 60% in gene expression in the sporophyte and gametophyte phases, supporting the concept that selection at the gametophytic level could be used to improve the performance of the sporophyte. !12'16~ The response ofsporophytes to various selective pressures such as temperature, salinity, heavy metals, and herbicides have been correlated with the sensitivity of gametophytes to the stressors./4/ For example, ZAMIR eta/. (17) reported that under low temperature stress, the male gametophyte from a cold-tolerant species was more competitive than the male gametophyte from a cold-sensitive species. SEARCY and MULCAHY (10) demonstrated that pollen grains from copper- or zinc-tolerant individuals ofSilene spp. and Mimulus guttatus were able to germinate and grow at concentrations of these heavy metals which would markedly inhibit pollen germination and tube growth in nontolerant selections. Generally, attempts at gametophytic selection have involved application of pollen to the stigma maintained in a stress environment to produce a selective pressure. This method, however, allows the pistil to participate in the selective process which, in some instances, appears to protect pollen from an environmental stress and prevent pollen selection. For example, SEARC'Z and MULCAnV/it) reported that pistils of copper-tolerant plants which contained enough of the metal to t To whom all correspondence should be addressed. 415 416 Y. HI ZHANG et al. inhibit the growth of pollen tubes in vitro did not inhibit pollen growth in vivo. KRAUSE eta/. <5>have reported that tube elongation of pollen from petunia (Petunia hybrida cv. White Bountiful) was retarded in vitro when treated with ozone, but not affected if deposited on stigmas and subsequently treated with ozone at 0.8/d/1. Since germinated and ungerminated pollen grains very likely differ in their haploid genetic contents, the ability to separate them would provide a method for locating stress-tolerant and -sensitive alleles. Previous attempts at segregating specific genotypes from a heterozygous pollen population, however, have not been entirely successful. Both BINO el al. (~> and MULCAHY et al. (81 employed centrifugation to separate germinated from ungerminated grains, but the efficiency of this separation method was low and only about 67% purity was obtained. In addition, the medium for density gradient centrifugation was toxic to the pollen./8~ In the present paper, a simple and effective method for separating germinated from ungerminated pollen grains is reported. MATERIALS AND M E T H O D S Pollen f~om a population of corn plants, Zea mays L. cv. Pioneer 3747, growing in 18-1 pots filled with a mixture of loam, peat, and sand (2:1 : l, v : v : v ) (one plant per pot) in a greenhouse with a minimum temperature of 18°C was used in this study. Supplemental light (using incandescent 400-W bulbs hanging 2 m above soil surface at a ratio of one bulb per 26 plants) was used from 1 November to 30 March to establish a 16-hr light condition per day to allow sufficient vegetative development before flowering. Vigorous growth of the plants was maintained by biweekly fertilization with 25 ml/pot of a 1% solution of water-soluble fertilizer (20-20-20, N : P205 : K~O). Fresh pollen was collected for each experimental trial by gently shaking tassels that had been vigorously shaken approximately 2 hr earlier to remove old pollen. (15) The pollen was cultured in a liquid medium based on that of CRAKER and WALDRON, (2) but modified by eliminating the agar and by increasing the sugar concentration to 20%. Pollen (4 mg) was placed on a sheet of weighing paper and distributed uniformly on the surface of culture medium (2 ml) in a Petri dish (3.5 cm in diameter) by gently shaking the pollen offthe weighing paper to the medium in the dish. Samples were cultured at 25°C for selected time intervals. Pollen germination and tube length were checked by sampling five different microscopic viewing fields in each Petri dish containing cultured pollen grains. Each treatment consisted of two replicate dishes. Pollen was considered germinated if the tube extrusion was greater than one half the diameter of the pollen grain. Pollen tube length was determined by measuring the three longest tubes in a microscopic viewing field (a total of 30 tubes/treatment) using a video monitor to trace the tubes and a SigmaScan software program to measure the length of tracings. <3>Pollen viability was assayed by reapplying separated pollen grains to culture medium for a measure of continued tube growth. Germinated were separated from ungerminated pollen grains using a screen and column system (Fig. 1). The column was packed with prewashed sea sand that could pass through a 16 mesh (Tyler screen scale) sieve, but not a 24 mesh sieve, a size range of sea sand that allowed the separation of germinated and ungerminated pollen grains in preliminary studies. Macrofiltration Plastic "7-i . tube Screen Liquid medium Sand column U Sponge Fro. 1. The screen and column system used to separate germinated and ungerminated pollen grains. The column is 6 cm long with a 0.87 cm inner diameter. The sand (24-16 mesh) is layered 4 cm high in the column. The sponge holds the sand particles in place and the plastic tube is added to keep the screen in place and guide the addition of culture medium used as the eluent. A M E T H O D T O SEPARATE POLLEN G R A I N S FIG. 2. Pollen grains cultured at 25°C for 1 hr. a: Before separation (mixture of germinated and ungerminated grains); b: after separation, from screen (germinated grains); c: after separation, from eluent (ungerminated grains). 417 A METHOD TO SEPARATE POLLEN GRAINS screens (Fisher Scientific) of three sizes having openings of 0.011 (0.105x0.105 mm), 0.022 (0.149x0.149 mm) and 0.044 (0.210x0.210 ram) mm% respectively, were tested. The column was filled with culture solution to above the level of the screen. For separation of germinated and ungerminated grains, the culture solution containing the mixture of germinated and ungerminated grains was layered on top of the solution in the column and the liquid phase in the system was immediately allowed to begin draining from the column. Approximately 21 ml of culture media solution was added in about 3-ml units during the draining process with a disposable Pasteur pipette to keep pollen grains in the solution suspended as the system drained. Germinated pollen grains caught on the upper surface of the screen were recovered by gently washing from the screen with culture media. Ungerminated grains passing through the screen and sand column were recovered by collections of the eluent. The mixture of ungerminated and germinated pollen grains passing through the screen but trapped in the sand column was discarded. 419 o O © v,,a RESULTS Germinated corn pollen grains could be effectively separated from ungerminated pollen grains. Successful separation required pollen germination and tube development for a minimum of 1 hr under our experimental conditions (Table 1). The separation process using the screen and column procedure resulted in an essentially pure •r. c-i 8 ~e ~Z O d O Table 1. Pollen separation tests Culture time (hr) Tube length* (mm) Mesh openingS" (mm2) Separation 0.5 1.0 2.0 0.290+0.008 0.700-t-0.018 1.385 _ 0.035 0.011 0.022 0.044 No Yes Yes * Mean __.S.E. ~Mesh opening smaller than 0.011 mm~ would retain large numbers ofungerminated pollen (diameter of corn pollen grain is approximately 0.1 mm). %-. © z O 420 Y. HI ZHANG 2.0 et al. minated pollen grains. BINOet al. (11 obtained 67% of germinated pollen grains in samples using low speed centrifugation. E 1.5 In the described procedure, the pollen grains always remain in non-toxic, culture media. Con~ 1.0 tinued and unretarded growth of tubes of the separated pollen grains demonstrates that the separation process causes no harm to the viability of 0.5 germinated pollen grains. In contrast, the toxicity to pollen of the centrifugation media used in the 0.0 density centrifugation is a serious problem. (8) Sample Sample Separated The mechanisms of separation using the screen sample at 1 h at 4 h at 4h column procedure are based on differences in density and size. As the pollen grains germinate, FIO. 3. Tube growth of pollen grains. Each sample the density decreases (8) and the pollen tubes consists of the cultured pollen grain population at the indicated time. The separated sample is germinated increase the physical size of the germinated grain. pollen grains recovered by the screen and column sys- During separation the ungerminated pollen tem and reapplied to culture media for continued tube grains sink to the screen, due to the pull of gravity and the density of the medium, before germinated growth. Mean+ S.E. grains and pass through the screen openings which are larger than the diameter of the pollen grains. When the germinated pollen grains with sample of germinated and ungerminated pollen tubes reach the screen, they are caught on the (Table 2, Fig. 2). The population of germinated screen surface as the length of the tube is larger pollen grains was contaminated by less than 1% than the opening in the screen. Germinated grains ungerminated pollen grains. The population of which because of their orientation pass through ungerminated pollen grains was contaminated the screen are trapped among the sand particles in by less than 30/0 germinated grains. Recovery of the column. Addition of medium solution during germinated pollen grains averaged 18.1 °/o of total draining appears to allow repetition of the sepgrains. Recovery of ungerminated pollen grains aration process, increasing the efficiency of separation. averaged 1.5% of total grains. The recovered, germinated pollen grains were The described separation technique is simple, viable as indicated by continued tube growth effective, and non-toxic to pollen. A separation of when reapplied to culture medium after sep- germinated from ungerminated pollen grains can aration; the tube growth of separated germinated be accomplished within 30 min, making segpollen grains was not retarded by the separation regated pollen readily available for use in further (Fig. 3). experimentation. For example, since post-meiotic gene expression in heterozygous plants generates stress-tolerant and -sensitive subpopulations of DISCUSSION pollen, the ability to quickly separate germinated The procedure described for separating ger- from ungerminated pollen grains would allow a minated pollen grains from ungerminated pollen method to recover stress-tolerant and -sensitive grains is effective and non-toxic. The purity of genotypes. The stress-tolerant and -sensitive subthe germinated pollen grains using the separation populations of pollen from a single segregating method averaged over 99%, much higher than plant should differ only in the locus (or loci and that obtained with other systems utilized for sep- closely linked loci) that determines the response arating germinated from ungerminated pollen to the stress, presenting what has been termed a grains. Previous tests by MULCAHY et al. (8) using "bulked segregant analysis" by MICHELMORE et density gradient centrifugation resulted in only a al. (6) Differences in the DNA of the sub66% separation of germinated from unger- populations of pollen would be useful in efforts to m m A M E T H O D TO SEPARATE POLLEN GRAINS o b t a i n seeds for stress-tolerant/sensitive plants and to detect genetic markers for stress-tolerant/ sensitive loci. Acknowledgments This study was supported (in part) from Experiment Station Project No. 499, Paper No. 3080, Massachusetts Agricultural Experiment Station, University of Massachusetts at Amherst. Thanks to Mr Zhi-Yi Tan for suggesting the screen used in separation of the pollen grains. 8. MULCAHY D. L., MULCAHY G. B., PoPP R., FONG N., PALLAISN., KALINOWSKIA. and MARIEN ,J. 9. 10. REFERENCES 1. BINO R. J., FRANKENJ. and VAN DER ZEEUW E. (1988) Method development for applying pollen selection in cucumber breeding programs: effects of centrifugation on pollen competence. Pages 2732 in M. CRESTI,P. GORI and E. PACINI,eds Sexual reproduction in higher plants. Springer, New York. 2. CRAKER L. E. and WALDRON P. F. (1989) Acid rain and seed yield reductions in corn. J. envir. Qual. 18, 127-129. 3. FEDER W. A. and SHRIER R. (1990) Combination of u.v.-B and ozone reduces pollen tube growth more than either stress alone. Envir. exp. Bot. 30, 451-454. 4. HORMAZAJ. I. and HERR~.RO M. 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