Pollen grains represent the male gametes of seed plants and their viability is critical for sexual reproduction in the plant life cycle. Palynology and viability studies have traditionally been used to address a range of botanical, ecological and geological questions, but recent work has revealed the importance of pollen viability in invasion biology as well. Here, we report an efficient visual method for assessing the viability of pollen using digital holographic microscopy (DHM). Imaging data reveal that quantitative phase information provided by the technique can be correlated with viability as indicated by the outcome of the colorimetric test. We successfully test this method on pollen grains of Lantana camara, a well-known alien invasive plant in the tropical world. Our results show that pollen viability may be assessed accurately without the usual staining procedure and suggest potential applications of the DHM methodology to a number of emerging areas in plant science.

The objective of this paper was to review the reproductive biology, herbicide-resistant (HR) biotypes, pollen-mediated gene flow (PMGF), and potential for transfer of alleles from HR to herbicide-susceptible grass weeds including barnyardgrass, creeping bentgrass, Italian ryegrass, johnsongrass, rigid (annual) ryegrass, and wild oats. The widespread occurrence of HR grass weeds is at least partly due to PMGF, particularly in obligate outcrossing species such as rigid ryegrass. Creeping bentgrass, a wind-pollinated turfgrass species, can efficiently disseminate herbicide resistance alleles via PMGF and movement of seeds and stolons. The genus Agrostis contains about 200 species, many of which are sexually compatible and produce naturally occurring hybrids and hybrids with species in the genus Polypogon. The self-incompatibility, extremely high outcrossing rate, and wind pollination in Italian ryegrass clearly point to PMGF as a major mechanism by which herbicide resistance alleles can spread across agricultural landscapes, resulting in abundant genetic variation within populations and low genetic differentiation among populations. Italian ryegrass can readily hybridize with perennial ryegrass and rigid ryegrass due to their similarity in chromosome numbers (2n = 14), resulting in interspecific gene exchange. Johnsongrass, barnyardgrass, and wild oats are self-pollinated species, so the potential for PMGF is relatively low and limited to short distances; however, seeds can easily shatter upon maturity before crop harvest, leading to wider dispersal. The occurrence of PMGF in reviewed grass weed species, even at a low rate, is greater than that of spontaneous mutations conferring herbicide resistance in weeds and thus can contribute to the spread of herbicide resistance alleles. This review indicates that the transfer of herbicide resistance alleles occurs under field conditions at varying levels depending on the grass weed species.

Pollen-mediated gene flow (PMGF) refers to the transfer of genetic information (alleles) from one plant to another compatible plant. With the evolution of herbicide-resistant (HR) weeds, PMGF plays an important role in the transfer of resistance alleles from HR to susceptible weeds; however, little attention is given to this topic. The objective of this work was to review reproductive biology, PMGF studies, and interspecific hybridization, as well as potential for herbicide resistance alleles to transfer in the economically important broadleaf weeds including common lambsquarters, giant ragweed, horseweed, kochia, Palmer amaranth, and waterhemp. The PMGF studies involving these species reveal that transfer of herbicide resistance alleles routinely occurs under field conditions and is influenced by several factors, such as reproductive biology, environment, and production practices. Interspecific hybridization studies within Amaranthus and Ambrosia spp. show that herbicide resistance allele transfer is possible between species of the same genus but at relatively low levels. The widespread occurrence of HR weed populations and high genetic diversity is at least partly due to PMGF, particularly in dioecious species such as Palmer amaranth and waterhemp compared with monoecious species such as common lambsquarters and horseweed. Prolific pollen production in giant ragweed contributes to PMGF. Kochia, a wind-pollinated species can efficiently disseminate herbicide resistance alleles via both PMGF and tumbleweed seed dispersal, resulting in widespread occurrence of multiple HR kochia populations. The findings from this review verify that intra- and interspecific gene flow can occur and, even at a low rate, could contribute to the rapid spread of herbicide resistance alleles. More research is needed to determine the role of PMGF in transferring multiple herbicide resistance alleles at the landscape level.

Kochia pollen dispersion was measured during 24 and 48 h periods from a kochia population in an 8- by 10-m area in the center of a 1.6 ha fallow field. Pollen counts from traps at 50- and 100-cm heights declined rapidly with increasing distance from the pollen source. Pollen deposition was highest along the prevailing wind direction: up to 23 pollen grains cm–2 were recovered 50 m from the pollen source along the southeast (SE) vector. Nonlinear regression analysis of pollen deposition along the SE vector was used to estimate that 99.9% of shed pollen would be deposited within 154.4 m of the source. Viability of pollen from greenhouse- and field-grown plants was measured using staining and germination assays. of four pollen stains tested, only 1,2,3-triphenyl tetrazolium chloride gave consistent results and did not stain heat-killed pollen. Depending on environmental conditions, kochia pollen remained viable from less than 1 d to 12 d. Length of kochia pollen viability was shortest under high temperatures (22 and 28 C) and low relative humidity (7 and 32%). Less than 0.5% germination was observed in 1.1% agar media with various additions; however, up to 17.8% germination was observed after incubation at 28 C in 100% relative humidity.

The movement of sulfonylurea herbicide-resistant (R) kochia pollen was investigated in a spring barley field near Moscow, ID, using a Nelder plot design in 1991 and 1992. Each 61 m diameter plot had 16 rays spaced 22.5° apart and contained 211 kochia plants. There were 12 susceptible (S) plants and one R plant along each ray. The R and S plants were 1.5 m and 3.0 to 30.5 m from the center of the plot, respectively. Wind direction and speed in the 16 vectors, air and soil temperature, and rainfall were monitored continuously. Mature kochia seed was collected from individual plants, planted in the greenhouse, and sprayed with chlorsulfuron to test for resistant F1 progeny. Results from the 2-yr study showed outcrossing of R pollen onto S plants at rates up to 13.1% per plant 1.5 m from the R plants and declining to 1.4% per plant or less 29 m from the R plants. At least 35% of the total R x S crosses occurred in the direction of prevailing southeastward winds. Predicted percentages of R x S crosses per plant ranged from 0.16 to 1.29 at 1.5 m, and 0.00 to 0.06% at 29 m. Thus, resistant kochia pollen can spread the sulfonylurea-resistant trait at least 30 m during each growing season.

Experiments were conducted in the North Carolina State University Phytotron greenhouse and field locations in Clayton, Rocky Mount, and Lewiston-Woodville, NC, in 2002 to determine the effect of glyphosate on pollen viability and seed set in glyphosate-resistant (GR) corn. Varieties representing both currently commercial GR corn events, GA21 and NK603, were used in phytotron and field studies. All glyphosate treatments were applied at 1.12 kg ai ha−1 at various growth stages. Regardless of hybrid, pollen viability was reduced in phytotron and field studies with glyphosate treatments applied at the V6 stage or later. Scanning electron microscopy of pollen from affected treatments showed distinct morphological alterations correlating with reduced pollen viability as determined by Alexander stain. Transmission electron microscopy showed pollen anatomy alterations including large vacuoles and lower starch accumulation with these same glyphosate treatments. Although pollen viability and pollen production were reduced in glyphosate treatments after V6, no effect on kernel set or yield was found among any of the reciprocal crosses in the phytotron or field studies. There were also no yield differences among any of the hand self-pollinated (nontreated male × nontreated female, etc.) crosses. Using enzyme-linked immunosorbent assay to examine CP4-5-enolpyruvlshikimate-3-phosphate synthase expression in DKC 64-10RR (NK603) at anthesis, we found the highest expression in pollen with progressively less in brace roots, ear leaf, anthers, roots, ovaries, silks, stem, flag leaf, and husk.

Greenhouse experiments were conducted to examine the effect of glyphosate on reproductive development in sicklepod. Glyphosate was applied postemergence over the top at 112 and 280 g ai/ha to sicklepod at 4-leaf stage (L), 8-L, 4-L followed by 8-L, and 12-L. A nontreated control was included. Immediately after the 12-L application, number of flowers was recorded for all treatments twice per week for 8 wk. Pollen viability was measured on 1 open flower/plant/sampling time using Alexander stain. The number of pods, pod length, seeds per plant, 50-seed weight, total seed weight, seed germination, seed viability, and dry weight of aboveground biomass were also recorded. No significant differences among the treatments were found for average pod length, 50-seed weight, seed germination, seed viability, and aboveground biomass. The nontreated had 18 flowers counted over 8 wk. Glyphosate applied at 12-L and sequentially at 4-L and 8-L, averaged over glyphosate rates, reduced cumulative flower production after 8 wk by 65 and 54%, respectively, compared with the nontreated. Similarly, glyphosate at 280 g/ha, averaged over treatment timings, reduced flower production by 58% compared with the nontreated. Because the number of flowers produced was limited by glyphosate treatment due to flower abscission, pollen viability measurements could not be analyzed because of large numbers of missing data points. The number of pods, seeds, and total seed weight were reduced by 79, 80, and 81%, respectively, with 280 g/ha of glyphosate compared with the nontreated.

Waterhemp is a major weed of field crops in the Midwestern United States. Its prevalence is at least partly due to its rapid evolution of resistance to many groups of herbicides over the last two decades. In light of its dioecy and anemophily, pollen movement in waterhemp is considered to be an important factor in the spread of herbicide resistance genes. Thus, the biology and dispersal profile of waterhemp pollen are critical determinants for understanding and predicting the spatial population dynamics of herbicide resistance in this species. In this study, pollen longevity was investigated with greenhouse experiments, and pollen dispersal and the effect of pollen competition were investigated in field plots. Pollen dispersal was determined by measuring the frequency of seeds produced on receptor plants positioned at various distances from a pollen source, which flowered in synchrony with the receptor plants. Results indicated that waterhemp pollen can remain viable up to 120 h, implying a low limitation of pollen dispersal by its longevity. Effective pollen dispersal declined exponentially with distance, with most pollen fertilizing recipient plants within 50 m of the pollen source. However, long distance pollen dispersal (800 m) was also observed. We also saw evidence for pollen swamping in this species. Under conditions of pollen competition among distinct genotypes, pollination success was inversely related to distance between pollen donors and receptors. However, relative pollen density may also play an important role in determining the rate of long distance gene flow. This study confirmed the potential of waterhemp pollen to effect long distance gene flow and provides supporting data for quantitative spatial modeling of waterhemp resistance dynamics.